U.S. patent application number 11/560308 was filed with the patent office on 2007-07-26 for molecular signaling pathways triggered by rituximab: prognostic, diagnostic, and therapeutic uses.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Benjamin Bonavida, Ali R. Jazirehi.
Application Number | 20070172847 11/560308 |
Document ID | / |
Family ID | 38285980 |
Filed Date | 2007-07-26 |
United States Patent
Application |
20070172847 |
Kind Code |
A1 |
Bonavida; Benjamin ; et
al. |
July 26, 2007 |
MOLECULAR SIGNALING PATHWAYS TRIGGERED BY RITUXIMAB: PROGNOSTIC,
DIAGNOSTIC, AND THERAPEUTIC USES
Abstract
The present invention provides markers associated with activated
molecular signaling pathways (example: p38 MAKP, NF-.kappa.B,
ERK1/2, YY-1 and AKT) inhibited by rituximab in cancer cells as
well as pathways activated by rituximab (such as death receptors,
RKIP, PTEN) all of which are associated with the regulation of
chemo and immunoresistance. The present invention provides methods
of prognosis and providing a prognosis for cancer such as lymphoma,
leukemia, and autoimmune disease, as well as, methods of drug
discovery. These markers are also therapeutic targets for treatment
of cancer resistant to conventional and experimental cancer
therapeutics. Inhibition or activation of expression and/or
activity of targeted gene products sensitizes resistant tumor cells
to subtoxic doses of cytotoxic treatment including chemotherapy,
radiation therapy, or immunotherapy and gene therapy, and the
cytotoxic molecules.
Inventors: |
Bonavida; Benjamin; (Los
Angeles, CA) ; Jazirehi; Ali R.; (Los Angeles,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
38285980 |
Appl. No.: |
11/560308 |
Filed: |
November 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60737301 |
Nov 15, 2005 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/6.16; 435/7.23 |
Current CPC
Class: |
G01N 33/5088 20130101;
G01N 33/57426 20130101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0001] This invention was made with Government support under
Department of Defense/US Army Grant DAMD 17-02-1-0023. The
Government has certain rights in this invention.
Claims
1. A method of diagnosing a cancer or providing a prognosis for
patient having a cancer that has altered expression of molecular
signaling pathways triggered by rituximab, the method comprising
the steps of: (a) contacting a tissue sample of the cancer with an
antibody that specifically binds to protein that is part of a
molecular signaling pathway triggered by rituximab; and (b)
determining whether or not expression of the protein is altered in
the sample, thereby diagnosing or providing the prognosis for the
cancer.
2. The method of claim 1, wherein the cancer is a CD20 expressing
cancer.
3. The method of claim 1, wherein the cancer is lymphoma.
4. The method of claim 1, wherein the molecular signaling pathway
is functional or activated AKT or NF.kappa.B.
5. The method of claim 1, wherein the protein is PTEN, AKT, Fas,
YY1, NF.kappa.B, NIK, IKK, or IKB.
6. The method of claim 1, wherein the protein is Bcl-2,
Bcl-.sub.XL, AP-1 or STAT3.
7. The method of claim 1, wherein the pathway is selected from a
p38 MapK/ Stat 3, Raf 1/MEK 1/2/ ERK 1/2, Nf.kappa.B or Akt
pathway.
8. The method of claim 4, wherein the tissue sample is at least one
of fixed or embedded in paraffin.
9. The method of claim 1, wherein the antibody is a monoclonal
antibody.
10. The method of claim 1, wherein the tissue sample is a
metastatic cancer tissue sample.
11. The method of claim 1, wherein the tissue sample is from blood,
bone marrow, prostate, ovary, bone, lymph node, liver, kidney, or
sites of metastases.
12. A method of diagnosing a cancer or providing a prognosis for a
patient having a cancer that that has altered expression of
molecular signaling pathways triggered by rituximab, the method
comprising the steps of: (a) contacting a tissue sample of the
cancer with a primer set of a first oligonucleotide and a second
oligonucleotide that each specifically hybridize to a nucleic acid
encoding a protein that is part of a molecular signaling pathway
triggered by rituximab; and (b) determining whether or not
expression of the nucleic acid is altered in the sample; thereby
diagnosing the cancer.
13. The method of claim 12, wherein amplifying YY1 nucleic acid is
amplified in the sample; and the expression of YY1 nucleic acid is
determined, wherein it is determined whether or not the cancer
overexpresses YY1.
14. A method of localizing a cancer that in vivo, the cancer having
altered expression of molecular signaling pathways triggered by
rituximab, the method comprising the step of imaging in a subject a
cell a polypeptide member of the molecular signaling pathways
triggered by rituximab, thereby localizing the cancer in vivo.
15. A method of identifying a compound that inhibits a cancer that
has an altered molecular signaling pathways triggered by rituximab,
the method comprising the steps of: (a) contacting a cell
expressing a polypeptide member of the molecular signaling pathways
triggered by rituximab with a compound; and (b) determining the
effect of the compound on the polypeptide; thereby identifying a
compound that inhibits the cancer.
16. A method of identifying a compound that inhibits a therapy
resistant cancer, the method comprising the steps of: (a)
contacting a cell expressing a polypeptide member of a molecular
signaling pathways triggered by rituximab with a compound; and (b)
determining the effect of the compound on the polypeptide; thereby
identifying a compound that inhibits the therapy resistant
cancer.
17. A method of treating or inhibiting a cancer in a subject that
that has an altered molecular signaling pathway triggered by
rituximab comprising administering to the subject a therapeutically
effective amount of one or more inhibitors that modulates a
polypeptide member of a molecular signaling pathway triggered by
rituximab.
18. The method of claim 17, wherein the inhibitor is a small
organic molecule or a chemical inhibitor.
19. The method of claim 17, wherein the inhibitor is an NO
donor.
20. The method of claim 18, wherein the NO donor is selected from
the group consisting of L-arginine, amyl nitrite, isoamyl nitrite,
nitroglycerin, isosorbide dinitrate, isosorbide-2-mononitrate,
isosorbide-5-mononitrate, erythrityl tetranitrate, pentaerythritol
tetranitrate, sodium nitroprusside, 3-morpholinosydnonimine,
molsidomine, N-hydroxyl-L-arginine, S,S-dinitrosodthiol, ethylene
glycol dinitrate, isopropyl nitrate, glyceryl-1-mononitrate,
glyceryl-1,2-dinitrate, glyceryl-1,3-dinitrate, glyceryl
trinitrate, butane-1,2,4-triol trinitrate,
N,O-diacetyl-N-hydroxy-4-chlorobenzenesulfonamide,
N.sup.G-hydroxy-L-arginine, hydroxyguanidine sulfate,
(.+-.)-S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione,
(.+-.)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide
(FK409),
(.+-.)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl]-3-pyridi-
necarboxamide (FR144420), 4-hydroxymethyl-3-furoxancarboxamide,
(Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate;
NOC-18; 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-* 1-triazene
(DETA/NONOate), NO gas, and mixtures thereof.
21. The method of claim 17, wherein the inhibitor is an siRNA.
22. The method of claim 17, wherein the inhibitor is an antimitotic
drug.
23. The method of claim 22, wherein the antimitotic drug is
selected from the group consisting of vinca alkaloids and
taxanes.
24. A method of treating or inhibiting a therapy resistant cancer
in a subject comprising administering to the subject a
therapeutically effective amount of one or more inhibitors of a
polypeptide member of a molecular signaling pathway triggered by
rituximab.
25. The method of claim 24, wherein the inhibitor is a small
organic molecule or a chemical inhibitor.
26. The method of claim 24, wherein the therapy resistant cancer
has altered expression of molecular signaling pathways triggered by
rituximab, and the therapy-resistant cancer was diagnosed by: (a)
contacting a tissue sample of the cancer with an antibody that
specifically binds to protein that is part of a molecular signaling
pathway triggered by rituximab; and (b) determining whether or not
expression of the protein is altered in the sample, thereby
diagnosing or providing the prognosis for the cancer.
27. The method of claim 24, wherein the one or more inhibitors are
administered concurrently with another cancer therapy.
28. A method of treating a patient having a CD-20 expressing
cancer, said method comprising administering to the patient a
modulator of a p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2, Nf-kappa B
or Akt pathway.
29. The method of claim 28, wherein the CD-20 expressing cancer is
selected from the group consisting of lymphoma, B-acute
lymphoblastic lymphoma, non-Hodgkin's lymphoma, Burkitt's small
cell, and large cell lymphomas, chronic lymphocytic leukemia,
Hodgkin's lymphoma, leukemia, acute myelogenous leukemia, acute
lymphoblastic leukemia, chronic modulator myelogenous leukemia, and
multiple myeloma.
30. The method of claim 28, wherein said modulator is a
pro-apoptosis modulator of a p38 MapK/ Stat 3, Raf 1/MEK 1/2/ ERK
1/2, Nf-kappa B or Akt pathway.
31. The method of claim 28, wherein said modulator binds PTEN, AKT,
Fas, YY1, NF.kappa.B, NIK, IKK, Bcl-2, Bcl-.sub.XL, AP-1, STAT3 or
IKB.
32. The method of claim 28, wherein the CD-20 expressing cancer was
identified to have a hyperactive p38 MapK/ Stat 3, Raf 1/MEK 1/2/
ERK 1/2, Nf-kappa B or Akt pathway.
33. The method of claim 32, wherein the CD-20 expressing cancer was
identified to have a hyperactive p38 MapK/ Stat 3, Raf 1/MEK 1/2/
ERK 1/2, Nf-kappa B or Akt pathway by determining whether or not
expression or amounts of a protein of the pathway is altered.
34. The method of claim 33, wherein the protein selected from the
group consisting of PTEN, AKT, Fas, YY1, NF.kappa.B, NIK, IKK,
Bcl-2, Bcl-.sub.XL, AP-1, STAT3, and IKB.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] Cancer is the second leading cause of death behind heart
disease. Cancer incidence and death figures account for about 10%
of the U.S. population in certain areas of the United States
(National Cancer Institute's Surveillance, Epidemiology, and End
Results (SEER) database and Bureau of the Census statistics; see,
Harrison's Principles of Internal Medicine, Kasper et al.,
16.sup.th ed., 2005, Chapter 66). The five leading causes of cancer
deaths among men are lung cancer, prostate cancer, colon and rectum
cancer, pancreatic cancer and leukemias. The five leading causes of
cancer deaths among women are lung cancer, breast cancer, colon
cancer, ovarian cancer and pancreatic cancer. When detected at
locally advanced or metastatic stages, no consistently curative
treatment regimen exists. Treatment for metastatic cancer includes
hormonal ablation, radiation therapy, chemotherapy, hormonal
therapy and combination therapies. Unfortunately, a resistance
often develops to further hormonal manipulation or to treatment
with conventional chemotherapy. Therefore, there is a need for
alternative therapies, such as immunotherapy or reversal of
resistance to chemotherapy, radiation therapy, and hormonal
therapy. For instance, immunotherapy is predicated on the notion
that all drug-resistant tumors should succumb to cytotoxic
lymphocyte-mediated killing. Such tumors may also develop
cross-resistance to apoptosis mediated by cytotoxic lymphocytes,
resulting ultimately in tumor progression and metastasis of the
resistant cells (Thompson, C., Science, 267:1456-62 (1995)). The
mechanism responsible for the apoptotic-resistant phenotype, if
identified, may be useful as a prognostic and/or diagnostic
indicator and may serve as a target for immunotherapeutic
intervention in the reversal of resistance to other cytotoxic
therapies.
[0004] The phosphatidylinositol 3-Kinase (PI3-K) is formed by
heterodimeric lipid kinases that catalyse the phosphorylation of
inositol-containing lipids, known as phosphatidylinositol (PtdIns),
allowing the conversion of phosphatidylinositol-3,4-biphosphate
(PtdIns-P2) to phosphatidylinositol-3,4,5-triphosphate (PtdIns-P3).
The latter is absent or undetectable in resting cells and although
PI3-K activity in normal cells is tightly regulated, it is
deregulated in a wide spectrum of tumors (Toker, A. et al., Cancer
Res., 66:3963-6 (2006); Noske, A. et al., Cancer letter, 11: [Epub
ahead of print] (2006); Guo, R. et al., J Steroid Biochem Mol
Biol., 99:9-18 (2006); Koul, D. et al., Mol Cancer Ther., 5:637-44
(2006); Liu, X. et al., Mol Cancer Ther., 5:494-501(2006)). Akt is
a serine/threonine protein kinase that mediates various downstream
effects of PI3-K. It plays a central role in signaling by the PI3-K
pathway by regulating many biological processes such as
proliferation, apoptosis and cell growth; moreover, it was
suggested to be involved in PI3-K-mediated tumorigenesis (Liu, X.
et al., Mol Cancer Ther., 5:494-501(2006); Castilla, C. et al.,
Endocrinology [Epub ahead of print] (2006)). The Akt pathway is of
particular interest because it regulates several critical cellular
functions, including cell cycle progression, migration, invasion,
and survival as well as angiogenesis. In addition, the activated
PI3K-Akt pathway provides major survival signals to lymphoma cells
and many other cancer cells (Toker, A. et al., Cancer Res.,
66:3963-6 (2006); Goswami, A. et al., Cancer Res., 66:2889-92
(2006)). Akt controls a variety of mechanisms that inhibit
apoptosis and prolong cell survival, exerting a positive effect on
NF-.kappa.B functions (Osaki, M. et al., Apoptosis, 9:667-76
(2004); Ozes, O. et al., Nature, 401:82-5 (1999)).
[0005] The lymphatic cancers known as non-Hodgkin's lymphoma (NHL)
are steadily increasing in prevalence worldwide. Although NHLs
initially respond to a variety of therapeutic modalities, they
exhibit an unremitting relapsing nature and are essentially
considered incurable. This pattern of inevitable failure of
standard therapies is due to the emergence of drug-resistant
variants which highlights the urgent need for the design of new
treatment regimens. Monoclonal antibodies (mAbs) targeted against
specific surface markers that are less systematically toxic and
less myelosuppressive, have provided an alternative therapeutic
approach.
[0006] About 80-85% of NHLs are of B-cell origin and .about.95% of
these express surface CD20 (1,2). One of the candidate antigens
that has been targeted for immunotherapy is CD20, a 297-amino acid
(32-37 kDa) unglycosylated phosphoprotein that spans the membrane
four times (Ernst, J. et al., Biochemistry, 44:15150-8 (2005)).
CD20 is a cell surface phosphoprotein that is expressed
specifically within the B-cell lineage from pre-B cells to mature B
cells. It is neither shed from the cell surface nor modulated or
internalized on antibody binding (Ernst, J. et al., Biochemistry,
44:15150-8 (2005)). Rituximab (chimeric anti-human CD20 antibody)
mediates its anti-tumor activity by multiple mechanisms that
include complement-dependent cytotoxicity (CDC), antibody-dependent
cellular cytotoxicity (ADCC) and induction of apoptosis following
CD20 cross-linking (Shan, D. et al., Blood, 91:1644-52 (1998);
Jazirehi, A. et al., Oncogene, 24:2121-43 (2005)). We have recently
reported that Rituximab sensitizes drug-resistant B Non-Hodgkin's
lymphoma (NHL) cell lines to the apoptotic effects of various
chemotherapeutic drugs via the selective downregulation of
Bcl-.sub.XL expression. Downregulation of Bcl-.sub.XL expression
was the result of inhibition of both the Raf/MEK/ERK1/2 and
NF-.kappa.B survival pathways (Jazirehi, A. et al., Cancer Res.,
64:7117-26 (2004); Jazirehi, A. et al., Cancer Res., 65:264-76
(2005)).
[0007] While rituximab has been successfully used in the treatment
of patients with non-Hodgkins lymphoma (NHL) its modes of action,
however, have not yet been fully elucidated. It has been reported
that the induction of antibody-dependent cell-mediated cytotoxicity
(ADCC), complement dependent cytoxicity (CDC), and seldom induction
of apoptosis may explain the efficacy of rituximab in vivo.
Supporting data for these mechanisms have been reported both in
vitro and in vivo and can be found in reviews (Smith, M., Oncogene,
22(47):7359-68 (2003); Jazirehi, A. et al., Oncogene,
24(13):2121-43 (2005)). The role of rituximab signaling in NHL
cells and its induced modification of intracellular survival
signaling pathways that regulate the proliferation states, the
expression of surface receptors and antiapoptotic pathways has not
been considered initially as potential mechanisms of rituximab
mediated effects. Further, the chemo sensitizing effect of
rituximab, initially reported by us (Demidem, A. et al., Cancer
Biother Radiopharm., 12(3):177-86 (1997); Alas, S. et al., Cancer
Res, 61:5137-44 (2001); Jazirehi, et al., 2003), and its underlying
molecular mechanisms were not examined. Alterations of cell
signaling upon administration of crosslinker (dimeric) ribuximab
has been reported following crosslinking of this antibody with a
secondary antibody (Deans, J. et al., J Immunol. 151(9):4494-504
(1993); Deans, J. et al., J Biol Chem. 270(38):22632-8 (1995)).
However, intracellular events triggered by monomeric rituximab was
not examined in these studies. The molecular signaling observed
following cross-linking are very distinct from the one reported in
this invention using monomeric rituximab.
[0008] Some NHL patients do not respond to rituximab treatment
alone and it is not clear why such patients are unresponsive. It
has been proposed that some of those patients exhibit polymorphism
in their Fc receptors expressed on their tumor cells, making such
cells resistant to ADCC (Cartron, G. et al., Blood, 99(3):754-8
(2002); Johnson, P. et al., Semin Oncol., 30(1 Suppl 2):3-8
(2003)). Thus, prior to the present invention, the state of art
with respect to signaling with monomeric rituximab was not known.
In this invention, several intracellular signaling pathways shown
to be modified by rituximab are further shown to be important in
the regulation of the tumor cell response to rituximab treatment
alone or in combination with chemotherapeutic drugs. This invention
identifies pathways that are modified by rituximab in which several
gene products regulate the response to apoptotic stimuli (e.g.,
chemotherapy, immunotherapy) following treatment with rituximab.
This invention also identifies gene products whose level of
expression may dictate tumor cells response to conventional
treatment. The invention therefore also identifies gene products
that are targets for therapeutic intervention. Failure of
chemotherapy to eliminate tumor cells has prompted the development
of alternative therapies.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides markers associated with
molecular signaling pathways such as functional or activated AKT,
NF.kappa.B, ERK 1/2, and p38MAPK that are triggered by rituximab in
CD-20 expressing cancer cells, including polypeptide members of the
pathways such as functional or activated Bcl-2/Bcl-.sub.XL, AKT,
PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB, and transcription
factors AP-1 and STAT3. These markers are therefore useful as
diagnostic and prognostic markers as well as for therapeutic
intervention targets (e.g., in drug assays and patient treatment).
The signaling pathways modified by rituximab are implicated in the
sensitization of tumor cells to death receptor mediated apoptotic
pathways (FasL, Trail, TNF.alpha.) cells and sensitize the cells to
cytotoxic immunotherapy (FASL, TNF, TRAIL) which are potent in
immunotherapeutic approaches to cancer treatment.
[0010] The chimeric mouse and human anti-CD-20 monoclonal antibody
rituximab (RITUXAN, IDEC-C2B8) has been approved by the FDA for the
treatment of B-Non Hodgkin's lymphoma (NHL). It has significant
anti-tumor activity and, alone or in combination with chemotherapy,
and has been successfully used in the treatment of patients with
follicular or low grade NHL (Czucman, M. et al., Semin. Oncol,
29:36-40 (2002)) and aggressive diffuse large B cell lymphoma
(DLBCL) in elderly patients (Coiffer, B., Semin Oncol, 30:21-7
(2003)). Rituximab treatment depletes CD20 positive normal and
cancerous B cells in patients. The postulated mechanisms of
rituximab-mediated effects include antibody dependent cellular
cytoxicity (ADCC), complement dependent cytotoxicity (CDC), and
induction of apoptosis (Maloney, D. et al., Semin. Oncol., 29:2-9
(2002); Smith, M., Oncogene, 22(47):7359-68 (2003); Jazirehi, A. et
al., Oncogene. 24(13):2121-43 (2005)). However, these postulated
mechanisms do not explain the failure of approximately 50% of NHL
patients to respond to rituximab treatment alone and do not explain
the enhanced response achieved with treatment combination of
rituximab and chemotherapeutic drugs in patients with
drug-resistant tumors.
[0011] This invention describes novel mechanisms of
rituximab-mediated activity which explain the underlying basis of
failure to respond to rituximab treatment alone and also explains
the molecular mechanism of rituximab-mediated sensitization to
chemotherapeutic drug-induced apoptosis in drug resistant B-NHL.
This invention describes the molecular signaling pathways triggered
by rituximab that result in the specific modifications of cell
survival signaling pathways utilized by the tumor cells and which
will result in the inhibition of cell proliferation and growth,
inhibition of gene products associated with resistance to apoptosis
and significant sensitization to a variety of chemotherapeutic
drugs. This invention also identifies a number of intracellular
gene products that are modified selectively by rituximab and which
are therefore molecular targets for the same indications as
rituximab. In addition, the signaling pathways modified by
rituximab in rituximab sensitive NHL cell lines identify gene
products whose over expression or otherwise modification or
mutation are involved in the resistance to rituximab mediated
affects. In addition, this invention can be utilized to evaluate
patient's tumors for a response or lack of response to rituximab
based on the profile of the signaling pathways modulated by
rituximab and thus has significant diagnostic/prognostic clinical
significance. While the above studies were performed in B-NHL cell
lines, the findings are applicable for other applications by
rituximab, currently under intensive investigations, in the
treatment of other B cell tumors and B cell mediated diseases such
as autoimmunity, rheumatoid arthritis, lupus, transplantation,
etc.
[0012] Generally, the methods find particular use in diagnosing or
providing a prognosis for cancer including prostate cancer, renal
cancer, lung cancer, ovarian cancer, breast cancer, colon cancer,
leukemias, B-cell lymphomas (e.g., non-Hodgkin's lymphomas,
including Burkitt's, small cell, and diffuse large cell lymphomas),
hepatocarcinoma or multiple myeloma. For example, these markers are
useful for profiling a cancer patient to determine their
sensitivity or resistance to rituxamib therapy, and for in vivo
imaging. In addition, the methods find use in drug assays for
cancer therapeutics, including the aforementioned cancers.
[0013] Accordingly, in one aspect the invention provides a method
of diagnosing a cancer or providing a prognosis for a cancer for a
patient that has altered expression of molecular signaling pathways
triggered by rituximab by determining whether or not expression or
amounts of a protein that is part of a molecular signaling pathway
triggered by rituximab is altered in a tissue sample of the cancer
from a patient, thereby diagnosing or providing the prognosis for
the cancer. In some embodiments, the tissue sample is contacted
with an antibody that specifically binds to protein that is part of
a molecular signaling pathway triggered by rituximab; and
determining whether or not expression of the protein is altered in
the sample, thereby diagnosing or providing the prognosis for the
cancer. In some embodiments, the cancer is a CD20 expressing
cancer, including lymphoma. The molecular signaling pathway can be
functional or activated AKT or NF.kappa.B. The protein can be PTEN,
AKT, Fas, YY1, NF.kappa.B, NIK, IKK, IKB, Bcl-2, Bcl-.sub.XL, AP-1
or STAT3 or other member set forth in FIGS. 5 or 11. In other
embodiments, the pathway is selected from a p38 MapK/Stat 3, Raf
1/MEK 1/2/ERK 1/2, Nf-kappa B or Akt pathway. The antibody can be a
monoclonal antibody. The tissue sample, in some embodiments, is one
fixed or embedded in paraffin. In yet other embodiments, the tissue
sample is a metastatic cancer tissue sample. The tissue sample, can
be from blood, bone marrow, prostate, ovary, bone, lymph node,
liver, kidney, or sites of metastases. In some embodiments of any
of the above, the methods indicates whether the cancer is sensitive
or resistant to rituximab or another agent (e.g., monoclonal
antibody) which binds CD20.
[0014] In still another aspect, the invention provides a method of
diagnosing a cancer or providing a prognosis for a cancer that that
has altered expression of molecular signaling pathways triggered by
rituximab, by contacting a tissue sample with a primer set of a
first oligonucleotide and a second oligonucleotide that each
specifically hybridize to a nucleic acid encoding a protein that is
part of a molecular signaling pathway triggered by rituximab; and
determining whether or not expression of the nucleic acid is
altered in the sample; thereby diagnosing the cancer; amplifying
YY1 nucleic acid in the sample; and determining whether or not YY1
nucleic acid is overexpressed in the sample; thereby diagnosing the
cancer that overexpresses YY1. In some embodiments of any of the
above, the cancer is a CD20 expressing or over-expressing cancer.
In other embodiments of any of the above, the cancer is a rituximab
resistant cancer.
[0015] In yet another aspect. the invention provides a method of
localizing a cancer in vivo, the cancer having altered expression
of molecular signaling pathways triggered by rituximab, imaging in
a subject a cell a polypeptide member of the molecular signaling
pathways triggered by rituximab (e.g., p38 MapK/ Stat 3, Raf 1/MEK
1/2/ERK 1/2, Nf-kappa B or Akt pathway), thereby localizing cancer
in vivo. The polypeptide member in some embodiments can be PTEN,
AKT, Fas, YY1, NF.kappa.B, NIK, IKK, IKB, Bcl-2, Bcl-.sub.XL, AP-1
or STAT3 or other member set forth in FIGS. 5 or 11. In some
embodiments the cancer is a CD20 expressing or over-expressing
cancer. In some embodiments of any of the above, the cancer is a
rituximab-resistant cancer.
[0016] In still other aspects, the invention provides methods of
identifying a compound or agent that inhibits a cancer that has an
altered molecular signaling pathways triggered by rituximab (e.g.,
a p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2, Nf-kappa B or Akt
pathway) by contacting a cell expressing a polypeptide member of
the molecular signaling pathways triggered by rituximab with a
compound; and determining the effect of the compound on the
polypeptide; thereby identifying a compound that inhibits the
cancer. The polypeptide member in some embodiments can be PTEN,
AKT, Fas, YY1, NF.kappa.B, NIK, IKK, IKB, Bcl-2, Bcl-.sub.XL, AP-1
or STAT3 or other member set forth in FIGS. 5 or 11. In some
embodiments the cancer is a CD20 expressing or over-expressing
cancer. In some embodiments of any of the above, the cancer is a
rituximab-resistant cancer. In some embodiments the cancer is a
CD20 expressing or over-expressing cancer.
[0017] In a related aspect, the invention provides methods of
identifying a compound that inhibits a therapy resistant cancer by
contacting a cell expressing a polypeptide member of a molecular
signaling pathways triggered by rituximab (see FIG. 5) with a
compound; and determining the effect of the compound on the
polypeptide; thereby identifying a compound as one that inhibits
the therapy resistant cancer. In some embodiments the cancer is a
CD20 expressing or under-expressing cancer. In some embodiments of
any of the above, the cancer is a rituximab-resistant cancer.
[0018] In a further aspect, the invention provides methods of
treating or inhibiting a cancer in a subject that that has an
altered molecular signaling pathway triggered by rituximab by
administering to the subject a therapeutically effective amount of
one or more inhibitors that modulates a polypeptide member of a
molecular signaling pathway triggered by rituximab(e.g., a p38
MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2, Nf-kappa B or Akt pathway).
The polypeptide member in some embodiments can be PTEN, AKT, Fas,
YY1, NF.kappa.B, NIK, IKK, IKB, Bcl-2, Bcl-.sub.XL, AP-1 or STAT3
or other member set forth in FIGS. 5 or 11. In some embodiments the
cancer is a CD20 expressing or over-expressing cancer. In some
embodiments of any of the above, the cancer is a
rituximab-resistant cancer. In some embodiments the cancer is a
CD20 expressing or over-expressing cancer. The inhibitor can be a
small organic molecule or a chemical inhibitor. In some
embodiments, the inhibitor is an NO donor. In still further
embodiments, the NO donor is selected from the group consisting of
L-arginine, amyl nitrite, isoamyl nitrite, nitroglycerin,
isosorbide dinitrate, isosorbide-2-mononitrate,
isosorbide-5-mononitrate, erythrityl tetranitrate, pentaerythritol
tetranitrate, sodium nitroprusside, 3 morpholinosydnonimine,
molsidomine, N-hydroxyl-L-arginine, S,S-dinitrosodthiol, ethylene
glycol dinitrate, isopropyl nitrate, glyceryl-1-mononitrate,
glyceryl-1,2-dinitrate, glyceryl-1,3-dinitrate, glyceryl
trinitrate, butane-1,2,4-triol trinitrate, N,O
diacetyl-N-hydroxy-4-chlorobenzenesulfonamide, NG
hydroxy-L-arginine, hydroxyguanidine sulfate,
(.+-.)-S-nitroso-N-acetylpenicillamine, S nitrosoglutathione,
(.+-.)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide
(FK409),
(.+-.)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl]-3-pyridi-
necarboxamide (FR144420), 4-hydroxymethyl-3-furoxancarboxamide,
(Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate;
NOC-18; 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-* 1-triazene
(DETA/NONOate), NO gas, and mixtures thereof. In other embodiments,
the inhibitor is an siRNA or an antimitotic drug (e.g., a vinca
alkaloid or taxane).
[0019] In yet other aspects, the invention provides methods of
treating or inhibiting a therapy resistant cancer in a subject
comprising administering to the subject a therapeutically effective
amount of one or more inhibitors of a polypeptide member of a
molecular signaling pathway triggered by rituximab. In an exemplary
embodiment, the cancer is a rituximab resistant cancer in which the
above survival pathways are hyper-activated and the inhibitors
reverse or oppose the drug-resistance by modulating a pathway
triggered by rituximab (see FIGS. 5., 11, and 13 to 15). The
inhibitor can be a small organic molecule or a chemical inhibitor.
In some embodiments, the therapy resistant cancer has altered
expression of molecular signaling pathways normally triggered by
rituximab, and the therapy-resistant cancer was diagnosed by
contacting a tissue sample of the cancer with an antibody that
specifically binds to protein that is part of a molecular signaling
pathway normally triggered by rituximab; and determining whether or
not expression of the protein is altered in the sample, thereby
diagnosing or providing the prognosis for the cancer. In some
embodiments, one or more inhibitors are administered concurrently
with another cancer therapy which may or may not include
rituximab.
[0020] In a still further aspect, the invention provides a method
of treating or chemo-sensitizing a patient having a CD-20
expressing cancer, said method comprising administering to the
patient a modulator of a p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2,
Nf-kappa B or Akt pathway or other rituximab-responsive
pathway(see, FIGS. 5, 11, and 13 to 15) or a polypeptide component
thereof. The CD-20 expressing cancer can be selected from the group
consisting of lymphoma, B-acute lymphoblastic lymphoma,
non-Hodgkin's lymphoma, Burkitt's small cell, and large cell
lymphomas, chronic lymphocytic leukemia, Hodgkin's lymphoma,
leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia,
chronic modulator myelogenous leukemia, and multiple myeloma. In
still further embodiments of any of the above, the modulator is a
pro-apoptosis or chemosensitizing modulator of a p38 MapK/ Stat 3,
Raf 1/MEK 1/2/ERK 1/2, Nf-kappa B or Akt pathway. In some
embodiments, the modulator binds PTEN, AKT, Fas, YY1, NF.kappa.B,
NIK, IKK, Bcl-2, Bcl-.sub.XL, AP-1, STAT3 or IKB. In some further
embodiments, the CD-20 expressing cancer was identified to have a
hyperactive p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2, Nf-kappa B or
Akt pathway. In yet other embodiments, the CD-20 expressing cancer
was identified to have a hyperactive p38 MapK/Stat 3, Raf 1/MEK
1/2/ERK 1/2, Nf-kappa B or Akt pathway by determining whether or
not expression or amounts of a protein of the pathway is altered.
In still further embodiments, the protein can be selected from the
group consisting of PTEN, AKT, Fas, YY1, NF.kappa.B, NIK, IKK,
Bcl-2, Bcl-.sub.XL, AP-1, STAT3, and IKB. In any embodiments of the
above, rituximab may or may not also be administered. In some
embodiments, the modulator binds to a marker selected from PTEN,
AKT, Fas, YY1, NF.kappa.B, NIK, IKK, Bcl-2, Bcl-.sub.XL, AP-1,
STAT3, and IKB.
[0021] Advantageously, the invention provides methods of
sensitizing cancers to chemotherapy or immunotherapy by
administering modulators of a p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK
1/2, Nf-.kappa.B or Akt pathway. Those are the p38 MapK/ Stat 3,
Raf 1/MEK 1/2/ERK 1/2, Nf-.kappa.B pathway and the Akt pathway.
Inhibition of these pathways provides selective inhibition
downstream of anti-apoptotic gene products such as Bcl-2 and
Bcl-.sub.XLand can result in the reversal of drug resistance and
chemo sensitize cancers to various chemotherapeutic drugs. These
inhibitors can mimic the effects of rituximab therapy and/or
chemosensitize tumor cells. They may be administered with or
without rituximab. The cancer can be CD-20 expressing cancer
selected from the group consisting of lymphoma, B-acute
lymphoblastic lymphoma, non-Hodgkin's lymphoma, Burkitt's small
cell, and large cell lymphomas, chronic lymphocytic leukemia,
Hodgkin's lymphoma, leukemia, acute myelogenous leukemia, acute
lymphoblastic leukemia, chronic modulator myelogenous leukemia, and
multiple myeloma. In still further embodiments of any of the above,
the modulator is a pro-apoptosis or chemosensitizing modulator of a
p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2, Nf-kappa B or Akt pathway.
In some embodiments, the modulator binds PTEN, AKT, Fas, YY1,
NF.kappa.B, NIK, IKK, Bcl-2, Bcl-.sub.XL, AP-1, STAT3 or IKB. In
some further embodiments, the CD-20 expressing cancer was
identified to have a hyperactive p38 MapK/ Stat 3, Raf 1/MEK
1/2/ERK 1/2, Nf-kappa B or Akt pathway. In yet other embodiments,
the CD-20 expressing cancer was identified to have a hyperactive
p38 MapK/Stat 3, Raf 1/MEK 1/2/ERK 1/2, Nf-kappa B or Akt pathway
by determining whether or not expression or amounts of a protein of
the pathway is altered. In still further embodiments, the protein
can be selected from the group consisting of PTEN, AKT, Fas, YY1,
NF.kappa.B, NIK, IKK, Bcl-2, Bcl-.sub.XL, AP-1, STAT3, and IKB. In
some embodiments, the modulator binds to a marker selected from
PTEN, AKT, Fas, YY1, NF.kappa.B, NIK, IKK, Bcl-2, Bcl-.sub.XL,
AP-1, STAT3, and IKB.
[0022] In another aspect, the invention provides a method of
immunotherapy for cancer cells expressing CD20 by administering
rituximab or another monoclonal antibody against CD20 with an
immunotherapeutic agent. Such treatment can regulate the cancer
cells' sensitivity to immunotherapy by upregulating death receptors
and sensitizing the cells to Fas ligand and TRAIL-induced
apoptosis. The upregulation of death receptors can result from the
inhibition of the transcription repressor Ying Yang 1 (YY 1) that
is itself regulated by Nf-.kappa.B. In addition, pharmacological
inhibitors for Nf.kappa.B or YY1 can be administered to provide a
similar therapeutic action as rituximab, with or without
co-administration of rituximab, in sensitizing tumor cells to
immunotherapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1. Inhibition of the PI3K/Akt signaling pathway in
Ramos by rituximab: (A) Ramos cells (1.times.10.sup.7/ml) were
treated with rituximab (20 .mu.g/ml) for different times (0-24 h)
and incubated at 37.degree. and cell lysates were prepared as
described in methods. The cell lysates were examined by western for
various unphosphorylated and phosphorylated (p) proteins of the Akt
pathway. B-actin was used as control for loading. (B) Inhibition of
the PI3K/Akt signaling pathway in Ramos by rituximab: Ramos cells
were treated with rituximab (138 nM) or with equal amount of
rituximab (Fab').sub.2 (78.3 n.mu.) for 20 h at 37.degree. and cell
lysates were prepared and examined for Akt and p-Akt (ser473).
B-actin was used as control. (C) Ramos cell lysates were prepared
as described above in A. and examined by western for
unphosphorylated and phosphorylated proteins of the NF-.kappa.B
pathway. B-actin was used as control. The above findings are
representative of three independent experiments yielding similar
results.
[0024] FIG. 2. Rituximab-mediated augmentation of the association
of Bad with Bcl-.sub.XL to form heterodimeric complexes: (A) Ramos
cells (2.times.10.sup.6/ml) were left untreated or were treated
with rituximab (20 .mu.g/ml) for 20 h and cell lysates were
prepared. The cell lysates were immunoprecipitated with Rabbit
anti-Bcl-.sub.XL antibody and the precipitate was examined by
western for Bad using Rabbit anti-Bad antibody as described in
Methods. The IgG bands in this figure correspond to the
immunoprecipitated rabbit anti-Bcl-.sub.XL IgG and its development
by the secondary goat anti-rabbit IgG and then developed with
HRP-goat anti-rabbit IgG. (B) Ramos cells were treated with
rituximab (20 .mu.g/ml), the Akt inhibitor LY294002 (25 .mu.m) or
with the combination for 24 h. The cell lysates were prepared and
examined by western for Bcl-.sub.XL levels. B-actin was used as
control. The above findings are representative of three independent
experiments yielding similar results.
[0025] FIG. 3. Chemosensitization of Ramos and Ramos RR1 by
rituximab, rituximab (Fab').sub.2 and by LY294002: (A) Ramos cells
were treated with different concentrations of rituximab for 20 h
and then treated with CDDP (15 .mu.g/ml) for an additional 24 h.
The cells were then evaluated for apoptosis by the PI method as
described in Methods. p<0.001 represents the combination
treatment of rituximab (0.138 .mu.m) and CDDP as compared to
treatment with CDDP alone. p<0.005 represents the combination
treatment of rituximab (0.104 .mu.M)+CDDP as compared to treatment
with CDDP alone. (B) Similar experiments to those described in A.
above were performed except that rituximab (Fab').sub.2 was used
instead of rituximab. p<0.001 represents the combination of
rituximab (Fab').sub.2 plus CDDP as compared to treatment with CDDP
alone. (C) Similar experiments as those described above in A. were
performed except that Ly294002 was used instead of rituximab.
p<0.005 represents the combination of LY294002 (20 and 30 .mu.M)
and CDDP as compared to treatment with CDDP alone. (D) Similar
experiments as those described above in (C) were preformed except
that adriamycin (ADR) was used instead of CDDP. p<0.001
represents the combination of LY294002 (10 and 25 .mu.M) plus ADR
(5 .mu.g/ml) as compared to treatment with ADR alone. (E) The
rituximab resistant Ramos clone, RR1, was treated as described
above in (A). for Ramos wild type. (F) The rituximab resistant
Ramos clone, Ramos RR1, was treated as described above in (C). for
Ramos wild type. p<0.005 represent the combination of LY294002
(60 .mu.M) and CDDP (15 .mu.g/ml) as compared to treatment with
CDDP alone. The above findings are representative of three
independent experiments yielding similar results.
[0026] FIG. 4. Direct role of the Akt pathway in chemosensitization
of Ramos to CDDP-induced apoptosis: (A) Ramos cells were
transfected with control siRNA or Akt siRNA for different periods
of time (0-72 h) as described in methods. Cell lysates were
prepared and examined by western for p-Akt, Akt and Bcl-.sub.XL
expression. (B) Ramos cells were transfected with control siRNA or
different concentrations of Akt siRNA for 48 h. The cells were then
treated with CDDP (15 .mu.g/ml) for an additional 25 h and the
cells were examined for apoptosis as described. p<0.001
represent the combinations of Ramos cells transfected with Akt
siRNA (5, 10, and 20 .mu.M) and CDDP as compared to treatment with
CDDP alone. The above findings are representative of three
independent experiments yielding similar results.
[0027] FIG. 5. Schematic diagram of rituximab-mediated inhibition
of the Akt pathway and chemosensitization: This diagram shows that
Ramos cells exhibit constitutively activated Akt and NF-.kappa.B
pathways and these are represented in dotted lines. Activation of
these pathways result downstream in cytosolic Bcl-.sub.XL that is
not complexed with p-Bad and overexpression of Bcl-.sub.XL leading
to chemoresistance. In contrast, treatment with rituximab inhibits
these pathways resulting in augmentation of association of
Bcl-.sub.XL and Bad as well as downregulation of Bcl-.sub.XL
expression leading to chemosensitization.
[0028] FIG. 6. (A) Surface expression of CD20 on RR1 clones. Cells
(2.times.10.sup.6) were stained with 1 .mu.g anti-CD20 mAb (IgG1
subtype; solid black lines) or isotype control (pure IgG1; gray
lines) and subsequently with FITC-labeled secondary Abs and
analyzed by FACS. The intensity of surface CD20 expression was
measured by the mean fluorescence intensity (MFI) (n=3). (B)
Rituximab inhibits the proliferation of the wt but not the RR
clones. Cells were left either untreated or treated with rituximab
(20 .mu.g/ml-24 h) and (10.sup.4 cells in triplicates) were used in
standard XTT assay. (C) An aliquot of the above samples were
examined microscopically to assess the ability of rituximab to form
homotypic aggregation of the clones. (D) Failure of cross-linked
rituximab to induce apoptosis in RR clones. Cells
(2.times.10.sup.6) were either left untreated or treated with
anti-hIg, rituximab or the optimal concentration of cross-linked
rituximab (50 .mu.g/ml anti-hIg+20 .mu.g/ml rituximab-24 h) and
subjected to PI assay for the percentage of apoptotic cells. (E)
Failure of rituximab to mediate CDC in the RR clones. Cells
(2.times.10.sup.6) were left either untreated or treated with human
Ab serum (5% and 10%), rituximab or the combination for 24 h and
subjected to PI staining (without fixing) for the percentage of
dead cells. (F) Failure of rituximab to chemo-sensitize the RR
clones. Cells (2.times.10.sup.6) were left either untreated or
pre-treated with rituximab (20 .mu.g/ml-24 h). Then, the cells were
washed, and were grown in complete medium supplemented with
paclitaxel (0.1-10 nM-18 h). Then, the cells were stained with PI
solution and apoptosis was assessed by FACS. Samples were set up in
duplicates and the results are represented as the mean.+-.SD (n=2).
*P values <0.05 significant compared to control.
[0029] FIG. 7. Higher drug-resistance and over-expression of the
anti-apoptotic Bcl-2 family members in the RR clones. A. Cells
(2.times.10.sup.6) were left either untreated or treated with
various concentrations of paclitaxel (1.0, 10, 20 .mu.g/ml), ADR
(0.5, 1.0, 2.0 .mu.g/ml), CDDP (1.0, 10, 20 .mu.g/ml), vincristine
(0.1, 0.5, 1.0 .mu.g/ml), and VP-16 (1.0, 10, 20 .mu.g/ml) for 18h.
Then, the cells were stained with PI solution and apoptosis was
assessed by FACS. Samples were set up in duplicates and the results
are represented as the mean.+-.SD (n=2). (Fold drug-resistance was
measured using the highest percentage of apoptosis induced by the
highest concentration of the drug in the wt cells as 100% and
calculating the required drug concentration to achieve the same
level of apoptosis in the clones) B. Total RNA of various culture
conditions was extracted from 10.sup.7 cells (as indicated) and
converted to cDNA. 2.5 .mu.g cDNA was used in qPCR analysis to
determine the transcripts levels. Levels of G-3-PDH were confirmed
for equal loading. Results are represented as mean.+-.SD of
triplicate samples. C. WCEs (40 .mu.g) of wt cells and RR clones
(.+-.20 .mu.g/ml rituximab-24 h) were subjected to immunoblotting
for protein levels. Levels of .beta.-actin were used for equal
loading (n=2). *P values <0.05 significant compared to
control.
[0030] FIG. 8. Hyper-activation of the NF-.kappa.B and ERK1/2
survival pathways in the RR clones. After overnight growth in
RPM11640+1% FBS, RR clones were washed and grown in complete
medium.+-.rituximab (20 .mu.g/ml; 24 h). A. Total cell lysates (40
.mu.g) were subjected to western blot analysis using
phospho-specific Abs for various components of the NF-.kappa.B and
ERK1/2 pathways. B. The kinase activity of the IKK complex using
I.kappa.B-.alpha. peptide (aa 1-50 including S.sup.32/36) and
ERK1/2 using MAPK kinase substrates 4 (aa 172-192) using
immune-complex kinase assay. C. After overnight growth in
RPMI1640+1% FBS, cells were washed and were grown in complete
medium (.+-.rituximab, DHMEQ, or PD098059). 10 .mu.g of nuclear
lysates were subjected to EMSA (n=2) (19, 20).
[0031] FIG. 9. Chemo-sensitization of the RR clones by chemical
inhibitors. Cells (2.times.10.sup.6) were left either untreated or
pretreated with A. DHMEQ (wt: 10 .mu.g/ml, clones: 20 .mu.g/ml), B.
bortezomib (wt: 4 .mu.M, clones: 8 .mu.M) or C. PD098059 (wt: 15
.mu.g/ml, clones: 30 .mu.g/ml) for 2 h. Cells were then incubated
with paclitaxel (1.0, 10, 20 .mu.g/ml), ADR (0.5, 1.0, 2.0
.mu.g/ml), CDDP (1.0, 10, 20 .mu.g/ml), vincristine (0.1, 0.5, 1.0
.mu.g/ml), and VP-16 (1.0, 10, 20 .mu.g/ml) for an additional 18h
and subjected to DNA fragmentation assay. The samples were set up
in duplicates and the results are represented as the mean.+-.SD of
two independent experiments. *P values <0.05 significant
compared to drug treatment alone.
[0032] FIG. 10. Inhibition of the expression of anti-apoptotic
factors by chemical inhibitors. RR1 clones were left either
untreated or treated with DHMEQ (20 .mu.g/ml), bortezomib (8.0
.mu.M) and PD098059 (30 .mu.g/ml). A. 2.5 .mu.g cDNA was used in
qPCR using gene specific primers. Levels of G-3-PDH were confirmed
for equal loading. Samples were set up in duplicates and the
results are represented as the mean.+-.SD (n=2). B. Total cell
lysates (40 .mu.g) were subjected to immunoblotting for Bcl-2,
Bcl-.sub.XL and Mcl-1. Levels of .beta.-actin were used for equal
loading (n=2). C. Role of anti-apoptotic Bcl-2 members in
chemo-sensitization. Cells were either left untreated or pretreated
with 2MAM-A3 (wt: 15 .mu.g/ml, clones: 35 .mu.g/ml-7 h). The cells
were then washed, treated with paclitaxel (10 nM-18 h) and
subjected to DNA fragmentation assay. Samples were set up in
duplicates and results are represented as mean.+-.SD of two
independent experiments. *P values <0.05 significant compared to
paclitaxel alone.
[0033] FIG. 11. Proposed model of the development of RR in NHL
B-cells. Among other mechanisms (18) alterations in the dynamics of
cell signaling, via an elusive mechanism, is a potential mechanism
for the development of resistance to rituximab therapy. Continuous
long-term rituximab exposure results in the development of NHL
B-clones that exhibit diminished CD20 surface expression. Compared
to the parental cells, the clones exhibit significantly lower
sensitivity to CDC-mediated killing, apoptosis induced by
cross-linked rituximab and higher levels of p-I.kappa.B-.alpha.,
p-IKK, p-ERK1/2 correlating with higher IKK and ERK1/2 kinase
activities leading to higher DBA of AP-1 and NF-.kappa.B
culminating in increased Bcl-2, Mcl-1, Bcl-.sub.XL expression.
Hyper-activation of these pathways will a) increase the
proliferation rate of the clones, b) increase the levels of Bcl-2,
Bcl-.sub.XL, Mcl-1 and the apoptosis threshold, and c) cause higher
chemo-resistance of the NHL B-clones. Specific pharmacological
inhibition of NF-.kappa.B and ERk1/2 pathways, or functional
impairment of anti-apoptotic Bcl-2 family members avert the
acquired chemo-resistance phenotype of the RR-clones inducing the
cells to undergo apoptosis in response to low levels of drugs.
[0034] FIG. 12. Chemosensitization of the RR clones by chemical
inhibitors. (A) RR clones (2.times.10.sup.6/treatment) were left
untreated or pretreated with DHMEQ (20 .mu.g/ml) bortezomib (8
.mu.M), or PD98059 (30 .mu.g/ml) for 2 hours. Cells were incubated
with paclitaxel (20 .mu.g/ml). ADR (2.0 .mu.g/ml), CDD_(20
.mu.g/ml) vincristine (1.0 .mu.g/ml) and VP-16 for an additional 18
hours and subjected to DNA fragmentation assay. Samples were set in
duplicates and the results represented as mean+/-SD of two
independent experiments. In all cases, statistically significant
values ( P<0.05) were obtained by the combination compared to
drug and/or inhibitor treatment alone. (B). Fold enhancement of
apoptosis by treatment of RR clones with inhibitors based on data
from part A.
[0035] FIG. 13. Roles of p38MAPK and STAT3 in the regulation of
Bcl-2 and chemoresistance in the 2F7 AIDS-derived DLBCL cell line.
This schematic diagram represents the effect of rituximab treatment
on inhibiting the activity of p38MAPK, NF-.kappa.B, SP1, IL-10
transcription and expression, IL-10-IL-10R signaling and STAT3
activity leading to downregulation of Bcl-2 and chemosensitization
of drug-resistant NHL cells.
[0036] FIG. 14. Riruximab-mediated inhibition of the ERK1/2,
NF-.kappa.B and AKT survival pathways in the Ramos and Daudi B-NHL
cell lines. This schematic diagram shows that rituximab inhibits
the Raf-1/Mek1/2/ERK1/2, NF-.kappa.B, and AKT signaling pathways
all leading to downregulation of the transcription and activity of
Bcl-xl and chemosensitization. In addition, rituximab induces RKIP
expression which participates in the inhibition of both the ERK1/2
and NFkB pathways. The rituximab resistant clones failed to respond
to rituximab treatment and no cell signaling was achieved and
therefore the tumor cell remained chemoresistant.
[0037] FIG. 15. Rituximab-mediated upregulation of death receptors
and sensitization to FasL and TRAIL-induced apoptosis in the 2F7,
Daudi and Ramos cell lines. This schematic diagram shows that
rituximab inhibits NF-.kappa.B and YY1 activities leading to
inhibition of the repressor activity of YY1 on both Fas and DR5
transcription and expression. This results in the upregulation of
these death receptors and sensitization of NHL cells to FasL and
TRAIL-induced apoptosis. In addition, pharmacologic inhibitors of
NF-.kappa.B and YY1 mimics rituximab and sensitizes the cells to
FasL and TRAIL-induced apoptosis.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The invention relates to the Applicants' discovery that
rituximab signals the lymphoma cells and inhibits several
intracellular signaling pathways (e.g., the p38 MapK/Stat 3, Raf
1/MEK1/2/ERK 1/2, Nf.kappa.B pathway and the Akt pathways) and that
pharmacological inhibitors of those various pathways can mimic
rituximab and chemosensitize tumor cells. Inhibition of these
pathways resulted in the selective inhibition downstream of
anti-apoptotic gene products such as Bcl-2 and Bcl-.sub.XL that
resulted in the reversal of drug resistance and chemo sensitized
the cells to various chemotherapeutic drugs. The invention also
relates to the Applicants' discoveries that upon the development of
rituximab resistance in their rituximab-resistant clones, the above
survival pathways are hyper-activated and that pharmacological
inhibitors of these pathways could reverse the drug-resistance.
Additionally, the Applicants have validated the cell lines examined
herein by demonstrating that in patients with non-Hodgkin's
Lymphoma the above signaling pathways are hyper-activated in the
examined tumor tissues.
[0039] The invention also relates to the Applicants' discovery that
rituximab treatment regulates the tumor cells sensitivity to
immunotherapy. The treatment resulted in upregulation of death
receptors and sensitization to Fas ligand and TRAIL-induced
apoptosis. The upregulation of death receptors by rituximab was the
result of the inhibition of the transcription repressor Ying Yang 1
that is itself regulated by Nf-.kappa.B. In addition,
pharmacological inhibitors for Nf.kappa.B or YY1 were found to
mimic rituximab and sensitize the tumor cells to immunotherapy.
Accordingly, the modulators of the survival pathways are
particularly useful in sensitizing a patient's cancer cells to both
chemotherapy and immunotherapy.
[0040] In particular, with respect to rituximab-mediated effects,
[0040] the following have been shown to be inhibited by rituximab:
[0041] Src kinases; p38 MAPK/STAT-3/IL-10/Bcl-2;
Ras/Raf-1/MEK1/2/ERK1/2/Bcl-x; NIK/IKK/IKB/TAK-1 /Bcl-x; PI3K/PDK1
/AKT/Bad/Bcl-xL; NF.kappa.B/YY1
[0042] In particular, with respect to rituximab-mediated effects,
the following was shown to be induced by rituximab: [0043] RKIP,
PTEN, Fas, DR5, Bad
[0044] In particular, with respect to rituximab-mediated effects,
the following transcription factors were shown to be inhibited by
rituximab: [0045] NF.kappa.B, AP-1, SP-1, STAT-3, YY1
[0046] Additionally, the following agents have been shown to
modulate markers and pathways. TABLE-US-00001 TABLE 2 Sensitizing
agents for drug-resistant B-NHL cell lines Sensitizing Modified
gene Chemotherapeutic Agent B-NHL cell line product(s) drug(s)
Reference Rituximab 2F7 DLBCL, Bcl-2 CDDP, (Alas, S. et al., 10C9
Fludarabine, ADR, Clinical Cancer Vinblastine Research, 7: 709-23
(2001)) Piceatannol 2F7 DLBCL Bcl-2 CDDP, (Alas, S. A. et
Fludarabine, ADR, al., Clin. Cancer Vinblastine Res. 9: 316-26
(2003)) Rituximab Raji, Daudi, Bcl-2, Bcl-.sub.xL Paclitaxel,
(Emmanouilides, Ramos, 2F7 Gemcitabine, C. et al., Cancer
Vinorelbine Biother and Radiopharm 17: 621-630 (2002)) Rituximab
Ramos, 2F7 Bcl-2, Bcl-.sub.xL, Paclitaxel, ADR, (Jazirehi, A. R.
Apaf-1 CDDP, Vincristine, et al., Molecular VP-16 Cancer Therapy,
2: 1183-93 (2003)) Rituximab, 2F7 p38 MAPK, NF-.kappa.B, CDDP
(Vega, M. I. et PP2, SB 203580, Sp-1, STAT-3, al., Oncogene, 23:
Bay 11-7085 IL-10, Bcl-2 3530-40 (2004)) Rituximab, GW Ramos, Daudi
Raf-1/MEK1/2/ Paclitaxel, ADR, (Jazirehi, A. R. 5074, PD- ERK1/2,
AP-1, CDDP, Vincristine, et al., Cancer 048059, UO126, Bcl-.sub.xL,
RKIP VP-16 Research, 64: 2MAM-A3 117-26 (2004)) Rituximab, Ramos,
Daudi p-NIK, IKK, I.kappa.B-.alpha., Paclitaxel, ADR, (Jazirehi, A.
R. Bay 11-7085, NF-.kappa.B, RKIP CDDP, Vincristine, et al., Cancer
DHMEQ, SN-50, VP = 16 Research 65: 2-MAM-A3 264-276 (2005))
Rituximab Ramos PI3K, PDK1, AKT, CDDP (Hongo, F. et al., IKK, Bad,
Bcl-.sub.xL, Biochemistry siRNA AKT and Biophysical Research
Communications 336: 692-701 (2005))
[0047] A number of approved drugs and antibodies can also interfere
with those pathways and those include: Gleevec, Bortezomib,
Trastuzumab, cetuximab/erbitux, gemtuzumab, doxil, gefitinib,
roferonA/intron-A, nitromed (NO donor for cardiovascular diseases),
prokine, avastin, campath and various chemotherapeutic drugs.
[0048] Accordingly, the above findings are of clear-cut prognostic,
diagnostic and therapeutic significance. Additionally, upregulation
of the death receptor by rituximab as well as the response of these
pathways to cytotoxic immunotherapy are significant
diagnostic/prognostic/and therapeutic targets.
[0049] Accordingly, this invention identifies molecular signaling
pathways (e.g., p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2,
Nf-.kappa.B pathway and the Akt pathways, and see FIG. 5, 11, 13 to
15) that can be modified following rituximab treatment of CD-20
expressing cancers such as NHL and that can segregate patients to
treatment with respect to therapy with rituximab alone or in
combination with chemotherapy. These pathways modified by rituximab
are intrinsically involved in the regulation of drug resistance and
identify several targets of therapeutic, prognostic and diagnostic
significance that are members of the signaling pathways, such as
functional or activated Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1,
NF.kappa.B, NIK, IKK, IKB, and transcription factors AP-1 and
STAT3. One way of practicing the invention is to examine patients'
tumor cells for the activation state of these signaling pathways so
as to determine the probability of whether they would be modified
by rituximab therapy alone or in combination with chemotherapy. For
example, patients' tumors that show hyper-activation of one or more
than one of these pathways and/or may have deficiencies in any of
the members of these pathways will be considered unlikely to
respond to treatment. Such patients will need a different
therapeutic approach and the use of either different drugs and/or
agents that can normalize the activity of the signaling pathways to
respond to treatment. Likewise, patients who become refractory to
treatment will also benefit from examining the tumor cells for the
status of these signaling pathways and their components, such as
functional or activated Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1,
NF.kappa.B, NIK, IKK, IKB, and transcription factors AP-1 and STAT3
and determine best courses for intervention. In addition, patients
with recurrences may be screened for the molecular signature of
these pathways (e.g., Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1,
NF.kappa.B, NIK, IKK, IKB, and transcription factors AP-1 and
STAT3) and then treated accordingly or by the corresponding
suitable treatment. The various analyses of the signaling pathways
may utilize tumor tissue for immunohistochemistry protein
expression by Western, transcription factor activity by EMSA,
transcripts by RT-PCR, microarray analysis, proteomics, etc. In
addition to the diagnostic/prognostic invention, this invention has
important therapeutic application and can be used to screen for
agents that can selectively modify aberrant abnormal signaling
pathways (functional or activated Bcl-2/Bcl-.sub.XL, AKT, PTEN,
Fas, YY1, NF.kappa.B, NIK, IKK, IKB, and transcription factors AP-1
and STAT3) in the cancer cells and that can then be used directly
or in combination with chemotherapy using conventional drugs.
[0050] This invention describes several signaling pathways and
their components (e.g., p38 MAPK pathway (SP1, STAT3, NF.kappa.B);
src/Raf1/ERK1/2 (MEK1/2, AP-1, Bcl-2/Bcl-.sub.XL,RKIP): NF.kappa.B
(NIK, IKK, IKB); AKT (PI3K, PDBK1, Bad, Bcl-.sub.XL, PTEN) (see
FIG. 5 also) triggered in NHL tumor cells following treatment with
rituximab which result in the modification of the activity of
specific cell signaling pathways that change the tumor cells
behavior and cell growth characteristics as well as reducing the
threshold for resistance to apoptotic stimuli. The pathways that
have been uncovered in this invention include the
Raf-1/MEK1/2/ERK1/2 and NF-.kappa.B, PTEN, P38MAPK signaling
pathways where rituximab treatment results in inhibition of their
activity. Further, this invention also describes a mechanism by
which rituximab is able to down regulate these pathways.
[0051] In one study (Jazirehi, A. et al., Cancer Res.
64(19):7117-26 (2004)) we demonstrate that treatment of B-NHL cell
lines with rituximab inhibited the kinase activity of mitogen
activated protein kinase (MEKK1/2) and reduced the phosphorylation
of the components of the ERK1/2 pathway (Raf-1/MEKK1/2/ERK1/2) and
decreased activation protein 1 (AP-1) DNA-binding activity
resulting in selective downregulation of the anti-apoptotic gene
product Bcl-.sub.XL. These above events occur with similar kinetics
and were observed 3 to 6 h following rituximab treatment. Rituximab
mediated affects were corroborated with specific pharmacological
inhibitors. In addition, the inhibition of the Raf-1/MEKK1/2/ERK1/2
pathway resulted in sensitization of the drug resistant tumor cells
to chemotherapeutic drug-induced apoptosis. In addition, we
demonstrated that rituximab induced the expression of Raf-1 kinase
inhibitor protein (RKIP) which was involved in the inhibition of
the ERK1/2 signaling pathway. These novel findings revealed a
signaling pathway modified by rituximab through the induction of
RKIP expression which adversely regulates the activity of the
ERK1/2 pathway and which regulates of Bcl-.sub.XL expression and
subsequently chemosensitization of drug refractory NHL tumor cells.
In addition, this study identifies several potential targets that
can be used for screening of new therapeutics and/or to modify
their expression in patients who are unresponsive to current
treatments. In addition, the pathways identified and modified by
rituximab may be used to identify patients tumor cells who might
not respond to rituximab treatment based on the level of expression
and/or activity.
[0052] In subsequent studies (Jazirehi, A. et al., Cancer Res.
65(1):264-76 (2005); Jazirehi et al., Abstract #288, Blood, 104(11)
(2004); Vega et al., Oncogene, 1-14 (2002); Vega et al., J.
Immunol., 175(4):2174-2183 (2005); Jazirehi et al., Cancer Res.,
65(1):264-276 (2005), herein incorporated by reference in their
entirety) we have reported the second novel signaling pathway
triggered by rituximab and modified following treatment of NHL cell
lines with rituximab. We demonstrated rituximab inhibits the
constitutively activated NF-.kappa.B activity and the NF.kappa.B
signaling pathway (including components functional or activated
Bcl-2/Bcl-.sub.XL, Fas, YY1, NF.kappa.B, NIK, IKK, IKB, and
transcription factors AP-1 and STAT3) leading to NF.kappa.B.
Rituximab decreased the phosphorylation of NF.kappa.B-inducing
kinase, I.kappa.B kinase, and I.kappa.B.alpha. and diminished IKK
kinase activity and decreased NF.kappa.B DNA-binding activity.
These events occurred rapidly following rituximab treatment (3 to 6
h). Rituximab induced Raf kinase inhibitor protein up-regulation
was in part responsible for interrupting the NF.kappa.B signaling
pathway and concomitant with down regulation of the anti-apoptotic
gene products Bcl-.sub.XL and Bfl/A1. Rituximab-mediated decreases
in the expression levels of Bfl-XL , Bfl/A1 were responsible for
rituximab mediated sensitization of drug resistance NHL cells to
various chemical therapeutic drugs-induced apoptosis. Therefore,
similar to the above findings, for the Raf 1/MEK1/2/ERK1/2
signaling pathway (modified by rituximab), the rituximab mediated
inhibition of the NF.kappa.B signaling pathway is also responsible
for patient response to rituximab treatment. In addition, it also
identifies a subset of patients whose pathway is overactive and
could not respond to rituximab treatment alone or in combination
with drugs.
[0053] Detection of members of the functional or activated a p38
MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2, Nf.kappa.B or Akt molecular
signaling pathways, such as functional or activated
Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB,
and transcription factors AP-1 and STAT3 is therefore useful for
diagnosis and prognosis of NHL as well as other CD-20 expressing
cancers, such as B-acute lymphoblastic lymphoma (B-ALL),
non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large
Cell lymphomas), chronic lymphocytic leukemia, and Hodgkin's
lymphoma, and multiple myeloma, as well as for B cell mediated
diseases such as autoimmune diseases, rheumatoid arthritis, lupus,
transplantation. Detection can include, for example, the level of
mRNA or protein expression, or the localization (i.e., in the
nucleus or the cytoplasm) of mRNA or protein. In terms of early
diagnosis, needle, surgical or bone marrow biopsies can be used and
examined by immunohistochemistry for expression in cytosol or
nuclei, alone or in combination with other markers such as p53,
usually negative in prostate cancer and other cancers. Thus, these
markers are a new positive stain that complements the traditional
negative stain to enhance the diagnosis of cancers. In addition,
microlaser microdissection can be used to isolate a few cells and
perform RT-PCR for nucleic acid detection. Molecular imaging can be
used to identify individual cells or groups of cells expressing
specific proteins (functional or activated Bcl-2/Bcl-.sub.XL, AKT,
PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB, and transcription
factors AP-1 and STAT3) or enzymatic activity in real time in
living patients (Louie et al., 2002). The value of imaging
systematically provides value for detection of metastatic cancer.
Finally, cells expressing functional or activated
Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB,
and transcription factors AP-1 and STAT3can be used for drug
discovery to identify new drugs to treat cancers, as well as to
evaluate cancer treatments. Such drugs can be directly used alone
or in combination with chemotherapy, immunotherapy, radiotherapy,
or hormonal therapy to treat cancers that are resistant to
therapy.
[0054] The invention provides methods for assaying for therapeutic
agents that inhibit of the p38 MapK/ Stat 3, Raf 1/MEK 1/2/ERK 1/2,
Nf-.kappa.B or Akt pathway signaling pathways, e.g., NF-KB inducing
kinase; IkB kinase (IKK); and IkB alpha. Methods of inhibiting
include decrease of NFkB DNA binding activity or a decrease of
Bcl-.sub.XL expression. The invention also provides methods of
identifying drug resistant tumors and chemosensitization drugs. For
example, inhibition of the NFkB pathway or Bcl-.sub.XL
expression/activity sensitizes drug resistant tumor cells to
chemotherapy-induced apoptosis. Prognostic/diagnostic markers
include hyperactivation of the components of the NFkB pathway and
overexpression of Bcl-.sub.XL. These correlate with tumor
progression and unresponsiveness to conventional therapeutics.
[0055] The invention provides therapeutic methods, including use of
rituximab in combination with immunotherapy: for example 1) agents
that activate the immune system to upregulate Fas L expression will
augment tumor cell response following rituximab treatment. 2)
administration of Fas ligand agonists or mimetics 3) administration
of antibody to Fas with no tissue toxicity 4) fusion of rituximab
with FasL or FasL like entity. The invention also provides means of
identifying agents that can inhibit YY1 expression/activity, such
as antisense, siRNA, small molecules, and NO donors. Inhibitors of
pathways that regulate YY1 will upregulate Fas on tumor cells and
sensitize them to host immune cells (without activation or
following activation with cytokines like interferon or IL2). Novel
therapeutics include a combination of rituximab and cytokines that
activate NK cells and upregulates surface FasL expression on NK
cells and enhance NK killing of the tumor cells. Inhibitors of the
NF-.kappa.B pathways also sensitize cells to Fas ligand, TRAIL, or
agonistic monoclonal antibodies to DR4 and DR5. Rituximab resistant
NHL may be treated with combination of agents that activate FasL on
host immune cells, alone or in combination with low doses of
sensitizing agents. All of the above for Fas-FasL can be applied
for TRAIL on effector cells and death receptors on tumor cells.
Data shows the roles of NFkB and YY2 in negatively regulating TRAIL
death receptors and resistance to TRAIL. Agents that can regulate
death receptors can be used on rituximab sensitive and rituximab
resistant tumor cells whereby the host immune cells will kill the
tumors. In addition, agents that upregulate FasL or TRAIL on host
cells can be used in combination. Resistance to rituximab can be
due in part to failure of host FasL/TRAIL to kill the tumor cells
due to defect in the expression of the receptors and their failure
to be upregulated by rituximab. For diagnostics and prognostics,
one can examine expression of FasL/TRAIL death receptors on tumor
cells. Low levels correlate with poor prognosis and may indicate
high risk for unresponsiveness to rituximab treatment. One can also
examine response to rituximab treatment for upregulation of death
receptors. If not, the prognosis is bad. One can also test for
overexpression/activity of YY1 in tumors. High levels correlates
with poor prognosis. One can test for sensitization to
FasL/TRAIL-induced apoptosis following rituximab treatment--failure
to respond indicates a poor prognosis/diagnosis. One can test for
circulating levels of death receptors prior or following treatment.
High levels show diagnostic/prognostic significance. Finally, one
can test for mutation of death receptors.
[0056] The present invention also demonstrates for the first time
that the mechanism underlying resistance of B-NHL to chemotherapy
may be distinguished from the mechanism of resistance to death
receptors. This translates in patients who are resistant to
chemotherapy or rituximab-mediated chemo-sensitization which may be
treated with immunotherapy using rituximab in combination with
immunomodulators of host immune system (e.g., natural killer cells
and macrophages are effector cells that can mediate ADCC.). The
present invention also identifies the NFkB pathway and Bcl-.sub.XL
expression responsible for chemoresistance and YY1 as responsible
for immune resistance. Inhibitors of NFkB pathway will sensitize
cells to both drug and immune mediated apoptosis. Inhibitors of
NFkB in combination with drugs/immunotherapy will recruit host
immune system to kill tumor cells. Clinical trial are being tested
with combination of rituximab and IL-2. The rationale is to
activate NK mediated ADCC. However, we provide other mechanisms by
which activated NK cells kill tumor cells by signaling death
receptors on the tumor cells. Overexpression of Bcl-.sub.XL
indicates chemoresistance and immune resistance. Overexpression or
hyperactivation of the NFkB pathway indicates resistance to both
chemo and immunotherapies. Overexpression of YY1 indicates immune
resistance. High levels of Bcl-xL/Bcl2 indicates poor prognosis.
Acccordingly, the expression levels of death receptors on tumor
cells can indicate failure of immunotherapy and a bad
prognosis.
[0057] The present invention also discloses that rituximab inhibits
the functional or activated AKT signaling pathway. Inhibition of
the functional or activated AKT pathways sensitizes cells to
drug-induced apoptosis. This provides the establishment of new
targets for intervention to reverse drug resistance. The invention
also provides drugs used clinically or in clinical trials that are
targeted at the functional or activated AKT pathway to act either
in combination with rituximab or in combination with other
chemotherapeutic drugs. Rituximab induces PTEN expression
(inhibitor of the AKT pathway). Agents that activate PTEN can be
used in the treatment of rituximab resistant NHL, alone or in
combination with conventional or experimental therapeutics.
Rituximab inhibits phosphorylation of Bad and increases the
Bad-Bcl-.sub.XL complex formation, thus reducing free Bcl-.sub.XL,
to act as anti apoptotic and a chemoresistance factor. The
invention therefore provides agents that can mimic rituximab in
combination with conventional therapeutics--development of
molecules that can inhibit phosphoBad or enhance binding of
Bcl-.sub.XL with Bad. Activation of the AKT pathway indicates a bad
prognosis and can also be determined by single analyses or
microarray analysis. Once can also determine the levels of PTEN
expression: which, if low, indicates a bad prognosis and resistance
to therapies. Circulating levels of members of the activated AKT
pathway or PTEN may predict recurrences or prognostic markers.
Cell Signaling and Chemosensitization
[0058] Our studies investigating the molecular signaling pathways
triggered by rituximab have resulted in the demonstration of
rituximab-mediated inhibition of the p38 MAPK, NF.kappa.B, ERK1/2,
and AKT signaling pathways. Inhibition of these pathways resulted
in the selective inhibition of Bcl-2/Bcl-.sub.XL expression leading
to chemosensitization and the reversal of drug resistance. Each of
the above signaling pathways affected by rituximab is described
briefly below.
[0059] a. Rituximab-mediated Inhibition of the p38 MAPK Signaling
Pathway
[0060] Using the DLBCL AIDS-derived B-NHL cell line 2F7 as a model,
which secretes cytokines such as TNF.alpha. and IL-10, we
demonstrate that rituximab disrupted selectively the IL-10
autocrine/paracrine loop and this correlated with both
downregulation of Bcl2 expression and chemosensitization. Rituximab
treatment did not inhibit other apoptotic regulatory proteins such
as Bcl-xL, BAD, p53, c-myc and latent membrane protein 1 (LMP1)
(Alas, S. et al., Clinical Cancer Research, 7:709-23 (2001)). The
signaling pathway by which rituximab decreases the transcription
and secretion of IL-10 in 2F7 was examined. We showed that
rituximab-mediated inhibition of IL-10 secretion resulted in
downregulation of constitutive STAT3 activity seen in those cells
(through IL-10-IL-10R interaction) and STAT3 inhibition resulted in
inhibition of Bcl2 transcription and expression. The direct role by
which rituximab-induced inhibition of STAT-3 activity and Bcl2
expression was corroborated by the use of a STAT 3 inhibitor
piceatannol (shown to inhibit the JAK1/Tyk-2-dependent STAT-3 and
STAT-5 signaling pathways) (Su, L. et al., The Journal of
Biological Chemistry, 275:12661-6 (2000)). Many studies have
reported that IL-10 is increased in the serum of many NHL patients
and that this increase correlates to a lower rate of survival
(Blay, J. Y. et al., Blood, 82:2169-74 (1993)). These studies
suggest a novel therapeutic strategy aiming at interfering with
IL-10 synthesis and secretion via inhibitors of the p38 MAPK/STAT-3
pathways.
[0061] In further studies, we examined the early events underlying
the molecular mechanism by which rituximab inhibits IL-10
transcription and secretion and resulting in inhibition of STAT-3
activity and Bcl-2 expression. We show that rituximab signals the
2F7 cells through the p38 MAPK pathway and results in the
inhibition of IL-1 0 transcription and secretion. Rituximab
inhibited the constitutive activity of Lyn and p38MAPK activities
resulting in inhibition of IL-10 transcription via inhibition of
SP-1. The role of p38MAPK in the regulation of IL-10 was
corroborated by the use of specific pharmacologic inhibitors of the
p38MAPK pathway and implicating the roles of Src kinases and
NF-.kappa.B. Rituximab-mediated inhibition of MAPK activity and
IL-10 transcription correlated with the inhibition of both STAT-3
activity and Bcl-2 expression and resulted in drug-induced
apoptosis.
[0062] We further examined the mechanism by which rituximab
inhibits Bcl-2 expression and sensitizes cells to drug-induced
apoptosis in 2F7 cells. We demonstrate that rituximab selectively
inhibits Bcl-2 expression with no effect on other examined
proteins. Treatment with CDDP induced the generation of
mitochondrial reactive oxygen species, specifically intra-cellular
peroxides. The combination of rituximab and CDDP acted
synergistically to induce apoptosis and mitochondrial mediated
apoptotic events (Alas, S. et al., Clinical Cancer Research,
8:836-845 (2002)).
[0063] b. Rituximab-mediated Inhibition of the Raf-1/MEK1/2/ERK1/2
Pathway.
[0064] The above studies performed in the DLBCL 2F7 cell line
following rituximab treatment did not address whether the same
effects are observed in the FL-NHL cell lines. Studies in Ramos and
Daudi revealed that rituximab selectively inhibited Bcl-.sub.XL and
leading to chemosensitization (Jazirehi, A. R. et al., Molecular
Cancer Therapy, 2:1183-93 (2003)). We examined the underlying
molecular mechanisms by which rituximab inhibits Bcl-.sub.xL
expression and demonstrate that rituximab inhibits the
Raf-1/MEK1/2/ERK1/2 AP-1 signaling pathway and downregulates
Bcl-.sub.xL expression which is under the transcriptional
regulation of AP-1. The ERK1/2 pathway is constitutively activated
in Ramos and Daudi and its inhibition by rituximab sensitizes the
cells to drug-induced apoptosis. The phosphorylation-dependent
state of Raf-1/MEK1/2/ERK1/2 was significantly decreased 3-6 h
post-rituximab treatment, concomitant with inhibition of MEK1/2
kinase ctivity. The role of the ERK1/2 pathway in the regulation of
Bcl-2 and chemosensitization was corroborated by the use of
specific pharmacologic inhibitors (GW-5074, PD-8098059, UO-126)
which also sensitize the cells to drug-induced apoptosis. In
addition, several lines of evidence corroborated the involvement of
the ERK1/2 pathway and the regulation of Bcl-xL expression. Bcl-xL
is abundantly expressed in lymphoma (Xerri, L. et al., British
Journal of Hematology, 92:900-6 (1996)) and protects the cells from
apoptosis induced by DNA-damaging agents. We have also analyzed
mechanisms that underlie rituximab-mediated inhibition of the
ERK1/2 pathway. Recently, Raf kinase inhibitor protein (RKIP) has
been identified as a negative regulator of the ERK1/2 pathway
(Yeung, K. et al., Nature, 401:173-7 (1999); Yeung, K. et al.,
Molecular Cellular Biology, 20:3079-85 (2000)). Therefore, we
examined whether RKIP induction by rituximab was involved in the
rituximab-induced inhibition of the ERK1/2 pathway. Our findings
reveal that rituximab upregulates the expression of RKIP and
facilitates the association of RKIP and Raf-1 (Jazirehi, A. R. et
al., Cancer Research, 64:117-26 (2004)). These findings unravel a
novel mechanism by which rituximab affects the ERK1/2 pathway and
inhibits downstream selectively Bcl-.sub.xL expression. This study
identified several potential targets for therapeutic intervention
(namely the components of the ERK1/2 pathway, Bcl-.sub.xL and RKIP)
and might provide a rational molecular basis for the therapeutic
use of inhibitors of the ERK1/2 pathway in combination with
chemotherapeutic drugs.
[0065] c. Rituximab-mediated Inhibition of the NF.kappa.B
Pathway.
[0066] The demonstration that rituximab inhibits selectively
Bcl-.sub.xL expression in NHL cell lines suggested that rituximab
may be inhibiting several signaling pathways that regulate
Bcl-.sub.xL expression. We have shown above that rituximab inhibits
the ERK1/2 pathway leading to downregulation of Bcl-.sub.xL
expression. Previous findings reported that NF.kappa.B also
regulates Bcl-.sub.xL gene expression (Ghosh, S. et al., Cell,
109:81-96 (2002); Dixit, V. et al., Cell, 111:615-9 (2002)).
Therefore, we examined whether rituximab modified the constitutive
activation of the NF.kappa.B pathway in Ramos and Daudi cell lines.
Indeed, we demonstrated that rituximab decreases the
phosphorylation of NF.kappa.B-inducing kinase, I.kappa.B kinase,
and I.kappa.B-.alpha. and diminishes IKK kinase activity and
decreases NF.kappa.B DNA-binding activity. In addition, rituximab
significantly upregulated RKIP expression, thus interrupting the
NF.kappa.B signaling pathway concomitant with Bcl-.sub.xL
downregulation. The role of NF.kappa.B inhibition in downregulation
of Bcl-.sub.xL and chemosensitization was corroborated by the use
of various inhibitors (Bay 11-7085, DHMEQ, SN-50). The induction of
RKIP expression augmented its physical association with endogenous
NIK, TAK-1 and IKK resulting in decreased activity of the
NF-.kappa.B pathway and diminishing NF-.kappa.B DNA-binding
activity and confirms the role of RKIP inhibition of NF-.kappa.B
(Yeung, K. C. et al., Molecular Cellular Biology, 21:7207-17
(2001)). These studies revealed another novel signaling pathway
triggered by rituximab and identified several potential targets for
therapeutic intervention (that is the components of the NF.kappa.B
pathway and RKIP). The findings also provide a rational molecular
basis for the use of rituximab or NF.kappa.B pharmacologic
inhibitors in combination with sub-toxic concentrations of
chemotherapeutic drugs in rituximab and drug-refractory NHL.
[0067] d. Rituximab-induced Inhibition of the AKT Signaling
Pathway.
[0068] As mentioned above, Bcl-.sub.xL expression is
transcriptionally regulated by various pathways and transcription
factors. The AKT pathway was reported to regulate Bcl-.sub.xL
activity and expression (Vivanco, I. et al., Nature Reviews Cancer
2:489-501 (2002)). We have recently found that, indeed, rituximab
treatment of Ramos cells inhibited the PI3K/AKT pathway, namely
inhibition of phospho PI3K, PDK-1, AKT with no effect on
non-phosphorylated proteins. In addition, inhibition of phospho-Bad
by rituximab augmented the association of Bad with Bcl-.sub.xL to
form complexes. In addition, inhibition of the AKT pathway also
inhibited the NF.kappa.B pathway and resulted in inhibition of
Bcl-.sub.xL expression as indicated above. The role of the AKT
pathway in the regulation of chemoresistance was corroborated by
the use of the AKT inhibitor Ly-294002 and by transfection with
siRNA AKT (Suzuki, E. et al., Proc Amer Assoc Cancer Res volume 47
(2006)). As described above for the p38 MAPK, ERK1/2 and
NF-.kappa.B pathways modified by rituximab, the present findings
revealed another pathway inhibited by rituximab and identifies the
AKT pathway as target for therapeutic intervention (see FIG.
14).
Chemosensitization of Drug-resistant B-NHL by Rituximab and
Pharmacological Inhibitors
[0069] We have described above various signaling pathways inhibited
by rituximab resulting in inhibition of apoptotic gene products and
reversal of drug resistance. These studies indicated that the
signaling pathways modified by rituximab are potential therapeutic
targets and whose intervention can mimic rituximab-mediated
chemosensitizing effects. Chemosensitization by rituximab and by
pharmacological inhibitors that were studied to-date by us and were
shown to reverse drug resistance in several NHL cell lines (namely
2F7, Raji Daudi and Ramos) are summarized in Table 1, farther
below.
Rituximab-induced Immunosensitization of B-NHL
[0070] The above findings on cell-signaling by rituximab, namely
inhibition of the survival signaling pathways (NF.kappa.B, ERK1/2,
p38 MAPK and AKT), resulted in significant inhibition of
anti-apoptotic gene products such as Bcl-2 and Bcl-.sub.xL, and
hence reversal of drug resistance. We have found that inhibition of
the transcription repressor Yin Yang 1 (YY1) by S-nitrosylation
(Hongo F. et al., Biochemistry and Biophysical Research
Communications 336:692-701 (2005)) or by siRNA YY1 (Huerta-Yepez,
S. et al., Oncogene 23:1993-5003 (2004)) resulted in upregulation
of Fas expression and sensitization to FasL-induced apoptosis in
ovarian and prostate carcinoma cell lines. We have shown that YY1
negatively regulates Fas transcription via its DNA-association to
the silencer region of the Fas promoter. Three potential YY1
responsive elements were found to cluster in a very narrow sequence
within the Fas promoter silencer region between -1619 and -1533 bp
relative to the transcription initiation site. Deletion of the
silencer region of the Fas promoter in a reporter assay resulted in
augmentation of Fas expression of the tumor cells (Garban, H. J. et
al., Journal of Immunology 167:75-81 (2001)). We have also reported
that YY1 is downstream of NF.kappa.B and is regulated by NF.kappa.B
activity. Since rituximab inhibited NF.kappa.B, we hypothesized
that rituximab may also inhibit YY1 and sensitizes NHL cell lines
to FasL-induced apoptosis. Indeed, our findings corroborated this
hypothesis and demonstrated that treatment of FasL (or CH-11
agonist monoclonal antibody) resistant B-NHL cell lines with
rituximab resulted in significant inhibition of YY1 expression and
activity, upregulation of Fas expression and sensitization to
CH-11-induced apoptosis. Fas expression was upregulated by
rituximab treatment as early as 6 h as determined by flow cytometry
for surface expression, reversed transcriptase polymerase chain
reaction for transcription, and Western blot for total protein.
Rituximab-induced inhibition of YY1 expression was determined by
Western and its DNA activity by EMSA. The involvement of NF.kappa.B
and YY1 in the regulation of Fas expression was corroborated by the
use of Ramos with a dominant active inhibitor of NF.kappa.B and by
silencing YY1 with YY1 siRNA, respectively. The role of
rituximab-mediated inhibition of the p38 MAPK/NF-.kappa.B/YY1
pathways in the regulation of Fas and sensitization to
CH-11-induced apoptosis was validated by the use of specific
pharmacological inhibitors of these pathways, all of which resulted
in sensitization to CH-11-induced apoptosis. The apoptotic pathway
involved in rituximab-mediated sensitization of NHL cells to
FasL-mediated apoptosis was examined. Treatment with rituximab
alone did not have any effect on the activation of caspases or on
the mitochondria. Likewise, treatment with CH-11 resulted in modest
activation of caspases 8 and 9, which correlated with moderate
induction of apoptosis. However, treatment with the combination
resulted in mitochondrial depolarization, release of cytochrome-C
and Smac/DIABLO, activation of caspases 9 and 3 and PARP cleavage,
suggesting the involvement of the Type II mitochondrial apoptotic
pathway (Barnhart, B. C. et al., Seminars in Immunology 15:185-93
(2003)). The activation of the mitochondrial pathway by combination
of rituximab and CH-11 may be the result of the inhibition of the
anti-apoptotic gene products Bcl-2/Bcl-.sub.xL by rituximab.
[0071] Accordingly, rituximab appears to exert a new mechanism of
action, namely the sensitization of tumor cells to host
FasL-induced apoptosis.
Roles of BCLxl and YY1 in Chemoresistance and Immune-resistance in
NHL, Respectively
[0072] Our findings clearly demonstrate that chemoresistance and
Fas resistance in NHL cell lines are commonly regulated by the
constitutive activation of NF.kappa.B. However, we demonstrate that
chemoresistance and Fas resistance are differentially regulated by
Bcl-.sub.xL and YY1, respectively. Rituximab-mediated inhibition of
NF.kappa.B activity resulted in both the inhibition of Bcl-.sub.xL
expression and chemosensitization and inhibition of YY 1 and
sensitization to CH-11-induced apoptosis. These differentially
regulated mechanisms for chemoresistance and immune-resistance
emanated from findings making use of biologically engineered cell
lines and specific pharmacological inhibitors. Treatment with
specific inhibitors for NF.kappa.B sensitized NHL cells to both
drug and CH-11-induced apoptosis. The role of Bcl-.sub.xL
expression in the regulation of drug resistance, but not Fas
resistance, was demonstrated by the failure of rituximab to
sensitize Bcl-.sub.xL overexpressing Ramos cells to drug-induced
apoptosis, although the same cells were still sensitive to
rituximab-induced sensitization to CH-11 apoptosis.
[0073] These findings clearly establish distinct regulatory
mechanisms modulated by rituximab in NHL cells downstream of
NF.kappa.B for the sensitization to Fas and drug-induced apoptosis.
This finding have clear clinical implications and suggest that
overexpression of Bcl-.sub.xL in tumors, which are refractory to
treatment with chemotherapeutic drugs, alone or in combination with
rituximab, may still be sensitive to killing by rituximab in
combination with immunotherapy (Vega, M. I. et al., Journal of
Immunology 175:2174-83 (2005)) (see FIG. 15). Accordingly, the
invention in one aspect provides methods of treating patients who
are ritixumab resistant by administering another agent which
modulates one or more components of a rituximab signal pathway in a
direction similar to that of rituximab to promote cancer cell
death.
Failure of Rituximab to Signal Rituximab-resistant NHL Clones
[0074] While rituximab used as monotherapy or in combination with
chemotherapy has improved significantly the treatment of patients
with NHL, there remains the problem of patients initially
unresponsive to rituximab and a subset of patients experiencing
unresponsiveness to further treatment. The mechanisms of
unresponsiveness have not been clear. It has been postulated that
CD20 downregulation (Kennedy, A. D. et al., Journal of Immunology
172:3280-8 (2004)), loss of CD20 (Haidar, J. H. et al., European
Journal of Hematology 70:330-2 (2003)), and circulating CD20
(Manshouri, T. et al., Blood 101:2507-13 (2003)) may be responsible
for resistance.
[0075] Based on our findings above with rituximab-mediated
inhibition of cell signaling, we find that the development of
rituximab resistance emanates from failure of rituximab to signal
the cells effectively, as well as the development of
hyper-activated survival signaling pathways and upregulation of
anti-apoptotic gene products. In order to test our hypothesis, we
have generated in vitro rituximab-resistant (RR) clones from Ramos,
Daudi, and 2F7 cells by culturing the cells in the presence of
increasing concentrations of rituximab for several weeks and
multiple cycles of limited dilutions.
[0076] We have examined representative clones (2F7RR1, Ramos RR1,
and Daudi RR1) for their response to rituximab as compared to
wildtype (wt) cells. Preliminary findings have been presented
(Jazirehi, A. R. et al., Blood 104:3410 (Abstract) (2004);
Jazirehi, A. R. et al., Blood 106:1514 (Abstract) (2005); Vega, M.
et al., Proc Amer Assoc Cancer Res 47. (2006)). Examination of
these clones revealed that they express some loss of CD20 on the
cell surface, were not responsive to complement-dependent
cytotoxicity, antibody-dependent cellular cytotoxicity, were not
growth-inhibited by rituximab, nor underwent apoptosis following
cross-linking of rituximab with a secondary anti-human IgG. We then
examined the cell-signaling mediated by rituximab on resistant
clones compared to wt. Preliminary findings demonstrate that in wt
Daudi cells, rituximab induces a rapid and transient increase in
A-SMase activity paralleled with cellular ceramide generation in
lipid rafts. In addition, rituximab treatment externalizes both
ceramide and A-SMase which co-localizes with the CD20 receptor
(Bezombes, C. et al., Blood, 104:1166-73 (2004)). In the resistant
clones, however, rituximab-induced A-SMase translocation and
ceramide generation at the cell surface was reduced (Jazirehi, A.
R. et al., Proc Amer Assoc Cancer Research 47 (2006)).
[0077] These findings indicate that the failure of rituximab to
mediate cell signaling may be due to failure of CD20 migration to
lipid rafts and initiating cell signaling. Further studies revealed
that rituximab failed to inhibit the p38 MAPK, ERK1/2 and
NF-.kappa.B signaling pathways. In addition, we have found that the
clones show hyper-activation of these pathways with overexpression
of Bcl-2/Bcl-.sub.xL. Noteworthy, the clones showed
cross-resistance to high concentrations of chemotherapeutic
drug-induced apoptosis compared to the response seen in wt. We then
examined if the RR clones' failure to be chemosensitized by
rituximab may be reverted to sensitivity. Our previous finding with
wt cells revealed that sensitizing agents that interfere with the
above signaling pathways modified by rituximab resulted in
significant chemosensitization that were comparable to rituximab.
Thus, we examined if such sensitizing agents can reverse the
resistance in the RR clones. Indeed, our findings established that
treatment of RR clones with inhibitors of the NF-.kappa.B pathway
(e.g. DHMEQ, Bortezomib, Bay 11-7085), the ERK1/2 pathway (e.g.
PD-098059), the p38 MAPK pathway (e.g. SB-203580) and Bcl-2 (e.g.
2MAM-A3) sensitized the resistant clones to various
chemotherapeutic drugs (e.g. Paclitaxel, Vincristine, VP-16, CDDP,
and Adriamycin).
[0078] These findings indicate that the acquired or development of
resistance of NHL cells to rituximab, alone or in combination with
chemotherapy, may still be amenable to treatment by combination of
sensitizing agents described above in combination with low doses of
conventional chemotherapy. In addition, these findings present
several new potential targets for the generation of new class of
inhibitors to reverse resistance. (See FIG. 14). Accordingly,
treatment with rituximab and immuno-modulating agents that
upregulate the expression of death ligands on host effector cells,
or co-administration of rituximab and TRAIL/anti-DR4/DR5, mAb can
be useful in treating cancer.
List of Abbreviations
[0079] ADCC: antibody-dependent cell-mediated cytotoxicity [0080]
AP-1: activator protein-1 [0081] ARL: acquired immunodeficiency
syndrome (AIDS)-related lymphoma [0082] Bcl-2: B cell lymphoma
protein 2 [0083] Bcl-.sub.XL: Bcl-2 related gene (long
alternatively spliced variant of Bcl-x gene) [0084] CDC:
complement-dependent cytotoxicity [0085] DBA: DNA-binding activity
[0086] DHMEQ: dehydroxymethylepoxyquinomicin [0087] DLBCL: diffuse
large B-cell lymphoma [0088] ERK1/2 MAPK: extracellular
signal-regulated kinase 1/2 mitogen activated protein kinase [0089]
IKK: inhibitor of kappa B (I.quadrature.B) kinase complex [0090]
FACS: fluorescence activated cell sorter [0091] Mcl-1: myeloid cell
differentiation 1 [0092] MDR: multi-drug resistance [0093] MEK1/2:
mitogen activated protein kinase kinase 1/2 [0094] 2MAM-A3:
2-methoxyantimycin-A3 [0095] NIK: nuclear factor .kappa.B
(NF-.kappa.B) inducing kinase [0096] PD098059:
[2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] [0097] RIPA:
radioimmuno-precipitation assay [0098] RKIP: Raf-1 kinase inhibitor
protein [0099] TRAIL: tumor necrosis factor (TNF)-related
apoptosis-inducing ligand [0100] XTT: sodium
3-[1-(phenylamino-carbonyl)-3, 4 tetrazolium]-bis
(4-metoxy-6-nitro) benzene Sulfonic acid hydrate Definitions
[0101] Markers for use according to the invention include gene
products that are modified by a rituximab triggered signaling
pathway disclosed herein. These markers include Bcl-2 family
members, surviving, IAPs, and cytokines. Exemplary markers include
"Functional or activated Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1,
NF.kappa.B, NIK, IKK, IKB, and transcription factors AP-1 and
STAT3." The recitals of markers refers to nucleic acids, e.g.,
gene, pre-mRNA, mRNA, and functional or activated polypeptides,
polymorphic variants, alleles, mutants, and interspecies homologs
that: (1) have an amino acid sequence that has greater than about
60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%,
preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater
amino acid sequence identity, preferably over a region of over a
region of at least about 25, 50, 100, 200, 500, 1000, or more amino
acids, to a polypeptide or a member of rituximab signaling pathway
disclosed herein, or modulated by a ritiuximab signaling pathway
disclosed herein or encoded by a referenced nucleic acid or an
amino acid sequence described herein; they include the human and wt
marker proteins (2) specifically bind to antibodies, e.g.,
polyclonal antibodies, raised against an immunogen comprising a
referenced amino acid sequence, immunogenic fragments thereof, and
conservatively modified variants thereof; (3) specifically
hybridize under stringent hybridization conditions to a nucleic
acid encoding a referenced amino acid sequence, and conservatively
modified variants thereof; (4) have a nucleic acid sequence that
has greater than about 95%, preferably greater than about 96%, 97%,
98%, 99%, or higher nucleotide sequence identity, preferably over a
region of at least about 25, 50, 100, 200, 500, 1000, or more
nucleotides, to a reference nucleic acid sequence. A polynucleotide
or polypeptide sequence is typically from a mammal including, but
not limited to, primate, e.g., human; rodent, e.g., rat, mouse,
hamster; cow, pig, horse, sheep, or any mammal. The nucleic acids
and proteins of the invention include both naturally occurring or
recombinant molecules. The genes and protein sequences for
functional or activated Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1,
NF.kappa.B, NIK, IKK, IKB, and transcription factors AP-1 and STAT3
and other members of the rituximab triggered signaling pathways
disclosed herein are well known in the art. Truncated and
alternatively spliced forms are included in the definition. In
addition, the definition includes phosphorylated forms and
enzymatically active or functional forms. Exemplary accession
numbers include the following: NP.sub.--644805, NP.sub.--001014432,
NP.sub.--000305, AAB35516, NP.sub.--003394, NP.sub.--003989,
BAA33714, AAD08996, AAD13528; AAC64675, and NP.sub.--0065390.
[0102] The phrase "molecular signaling pathways triggered by
rituximab in cancer cells, including functional or activated A
Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB,
and transcription factors AP-1 and STAT3 refers to pathways
disclosed herein, including NF.kappa.B and AKT, and polypeptide
members thereof known in the art.
[0103] "Functional or activated" refers to the altered activation
state of molecular pathways triggered by rituximab. The studies
described herein show that rituximab alters the activation state of
the survival signaling pathways (e.g., ERK1/2; NFkB, p38MAPK, AKT).
The activation state is determined by phosphorylation and enzymatic
activity. The basal non phosphorylated protein levels are not
altered by rituximab. In the resistant clones these activated
signaling pathways are not inhibited by rituximab and particularly
are hyperactivated.
[0104] "Cancer" refers to human cancers and carcinomas, sarcomas,
adenocarcinomas, lymphomas, leukemias, chronic lymphocytic
leukemia, etc., including solid and lymphoid cancers, kidney,
breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach,
brain, head and neck, skin, uterine, testicular, glioma, esophagus,
and liver cancer, including hepatocarcinoma, lymphoma, including
B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g.,
Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's
lymphoma, leukemia (including AML, ALL, and CML), and multiple
myeloma. The invention is useful for any cancer or cell that
expresses CD-20 such as lymphoma, including B-acute lymphoblastic
lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and
Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including
AML, ALL, and CML), chronic lymphocytic leukemia, and multiple
myeloma, as well as for B cell mediated diseases such as autoimmune
diseases, rheumatoid arthritis, lupus, transplantation, etc.
[0105] "Therapy resistant" or "chemo-resistant" cancers, tumor
cells, and tumors refers to cancers that have become resistant to
both apoptosis-mediated (e.g., through death receptor cell
signaling, for example, Fas ligand receptor, TRAIL receptors,
TNF-R1) and non-apoptosis mediated (e.g., antimetabolites,
anti-angiogenic, etc.) cancer therapies. "Therapy sensitive"
cancers are not resistant to therapy. Cancer therapies include
chemotherapy, hormonal therapy, radiotherapy, gene therapy, and
immunotherapy (e.g., vaccines). Such therapies include
administration of chemotherapeutic drugs, antibodies, immunotoxins,
proteasome inhibitors, or chemical inhibitors. The agents can
mediate both apoptosis and non apoptosis mediated cytotoxicity.
[0106] In a preferred embodiment, the methods of the invention can
be used to evaluate or diagnose whether a cancer is resistant with
respect to a variety of anticancer agents that induce or stimulate
apoptosis and inform the selection of a therapy avoiding or
opposing the mechanism of resistance. These include, but are not
limited to, radiation (e.g., X-rays, gamma rays, UV); tumor
necrosis factor (TNF)-related factors (e.g., TNF family receptor
proteins, TNF family ligands, TRAIL, antibodies to TRAILR1 or
TRAILR2); kinase inhibitors (e.g., epidermal growth factor receptor
(EGFR) kinase inhibitor, vascular growth factor receptor (VGFR)
kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase
inhibitor, platelet-derived growth factor receptor (PDGFR) kinase
inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC));
antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN,
and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen);
anti-androgens (e.g., flutamide, bicalutamide, finasteride,
aminoglutethamide, ketoconazole, and corticosteroids);
cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam,
NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs));
anti-inflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE,
dexamethasone, dexamethasone intensol, DEXONE, HEXADROL,
hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone,
PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone,
PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g.,
irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine
(DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin,
carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine,
bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular
signaling molecules; ceramides and cytokines; staurosporine, and
the like.
[0107] In still other embodiments, the proapoptitic agents include
anti-hyperproliferative or antineoplastic agents selected from
alkylating agents, and antimetabolites.
[0108] Alkylating agents can be 1) nitrogen mustards (e.g.,
mechlorethamine, cyclophosphamide, ifosfamide, melphalan
(L-sarcolysin); and chlorambucil); 2) ethylenimines and
methylmelamines (e.g., hexamethylmelamine and thiotepa); 3) alkyl
sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine
(BCNU); lomustine (CCNU); semustine (methyl-CCNU); and streptozocin
(streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC;
dimethyltriazenoimid-azolecarboxamide).
[0109] In some embodiments, the antimetabolites can be 1) folic
acid analogs (e.g., methotrexate (amethopterin)); 2) pyrimidine
analogs (e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine
(fluorode-oxyuridine; FudR), and cytarabine (cytosine
arabinoside)); and 3) purine analogs (e.g., mercaptopurine
(6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and
pentostatin (2'-deoxycoformycin)).
[0110] In still further embodiments, the pro-apoptosis and/or
chemotherapeutic agents can be 1) vinca alkaloids (e.g.,
vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g.,
etoposide and teniposide); 3) antibiotics (e.g., dactinomycin
(actinomycin D), daunorubicin (daunomycin; rubidomycin),
doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin
(mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological
response modifiers (e.g., interferon-alfa); 6) platinum
coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin);
7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas
(e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g.,
procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical
suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11)
adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,
hydroxyprogesterone caproate, medroxyprogesterone acetate, and
megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and
ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15)
androgens (e.g., testosterone propionate and fluoxymesterone); 16)
antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing
hormone analogs (e.g., leuprolide).
[0111] Resistance to any oncolytic agent that is routinely used in
a cancer therapy may be evaluated by assessing the state of the
rituximab-triggered pathways and their components. For example, the
U.S. Food and Drug Administration maintains a formulary of
oncolytic agents approved for use in the United States.
International counterpart agencies to the U.S.F.D.A. maintain
similar formularies. Table 1 provides a list of exemplary
antineoplastic agents approved for use in the U.S. Those skilled in
the art will appreciate that the "product labels" required on all
U.S. approved chemotherapeutics describe approved indications,
dosing information, toxicity data, and the like, for the exemplary
agents. Listed agents include aldesleukin, Alemtuzumab,
allopurinol, arsenic trioxide, asparaginase azacitidine,
bevacizumab, bexarotene, bortezomib, busulfan, capecitabine,
carboplatin, carboplatin, carmustine, cetuximab, chlorambucil,
cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, actinomycin D, dasatinib, daunomycin,
decitabine, denileukin, diftitox, , docetaxel, doxorubicin,
erlotinib, etoposide phosphate, floxuridine, fludarabine,
5-fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab
ozogamicin, goserelin acetate, goserelin, histrelin acetate,
Ibritumomab Tiuxetan, idarubicin ifosfamide, imatinib mesylate
Interferon alfa-2a, Interferon alfa-2b, irinotecan, lenalidomide,
letrozole, leucovorin, Leuprolide, levamisole, lomustine, CCNU,
meclorethamine, nitrogen mustard, megestrol acetate, melphalan,
L-PAM, 6-mercaptopurine, methotrexate, methoxsalen, mitomycin C,
mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine,
nofetumomab, oxaliplatin, paclitaxel, pamidronate, pegaspargase,
pentostatin, pipobroman, plicamycin, mithramycin, procarbazine,
quinacrine, Rituximab, sorafenib, streptozocin, sunitinib maleate,
tamoxifen, temozolomide, teniposide, VM-26, testolactone,
thalidomide, 6-thioguanine, thiotepa, topotecan, toremifene,
Tositumomab, Trastuzumab, tretinoin, ATRA, Uracil Mustard,
valrubicin, vinblastine, vincristine, vinorelbine.
[0112] For a more detailed description of anticancer agents and
other therapeutic agents, those skilled in the art are referred to
any number of instructive manuals including, but not limited to,
the Physician's Desk Reference and to Goodman and Gilman's
"Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman
et al., 2002.
[0113] "Therapy-mediated or induced cytotoxicity" refers to all
mechanisms by which cancer therapies kill or inhibit cancer cells,
including but not limited to inhibition of proliferation,
inhibition of angiogenesis, and cell death due to, for example,
activation of apoptosis pathways (e.g., death receptor cell
signaling, for example, Fas ligand receptor, TRAIL receptors,
TNF-R1). Cancer therapies include chemotherapy, immunotherapy,
radiotherapy, and hormonal therapy.
[0114] "Therapeutic treatment" and "cancer therapies" and "cancer
therapy reagents" refers to apoptosis-mediated and non-apoptosis
mediated cancer therapies that treat, prevent, or inhibit cancers,
including chemotherapy, hormonal therapy (e.g., androgens,
estrogens, antiestrogens (tamoxifen), progestins, thyroid hormones
and adrenal cortical compounds), radiotherapy, and immunotherapy
(e.g., ZEVALIN, BEXXAR, RITUXAN (rituximab), HERCEPTIN). Cancer
therapies can be enhanced by administration with a sensitizing
agent, as described herein, either before or with the cancer
therapy.
[0115] "Chemotherapeutic drugs" include conventional
chemotherapeutic reagents such as alkylating agents,
anti-metabolites, anti-mitototics, plant alkaloids, antibiotics,
and miscellaneous compounds e.g., cis-platinum, CDDP, methotrexate,
vincristine, adriamycin, bleomycin, and hydroxyurea.
Chemotherapeutic drugs also include proteasome inhibitors such as
salinosporamides, bortezomib, PS-519, and omuralide. The drugs can
be administered alone or combination ("combination
chemotherapy").
[0116] By "sensitizingly effective amount or dose" or
"sensitizingly sufficient amount or dose" herein is meant a dose or
a modulator or agent that produces cancer cell sensitizing effects
for which it is administered. The exact dose will depend on the
purpose of the treatment, and will be ascertainable by one skilled
in the art using known techniques (see, e.g., Lieberman,
Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art,
Science and Technology of Pharmaceutical Compounding (1999);
Pickar, Dosage Calculations (1999); and Remington: The Science and
Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott,
Williams & Wilkins). Sensitized cancer cells respond better to
cancer therapy (are inhibited or killed faster or more often) than
non-sensitized cells, as follows: Control samples (untreated with
sensitizing agents) are assigned a relative cancer therapy response
value of 100%. Sensitization is achieved when the cancer therapy
response value relative to the control is about 110% or 120%,
preferably 200%, more preferably 500-1000% or more, i.e., at least
about 10% more cells are killed or inhibited, or the cells are
killed or inhibited at least about 10% faster. Cancer therapy
response value refers to the amount of killing or inhibition of a
cancer cell, or the speed of killing or inhibition of a cancer cell
when it is treated with a cancer therapy. Some compounds are useful
both as therapeutic reagents and as sensitizing reagents. Often, a
lower dose (i.e., lower than the conventional therapeutic dose) or
sub-toxic dose of such a reagent can be used to sensitize a cell.
Often, when a cell is sensitized, a lower dose of the
chemotherapeutic reagent can be used to achieve the same
therapeutic effect as with a cell that has not been sensitized.
[0117] By "therapeutically effective amount or dose" or
"therapeutically sufficient amount or dose" herein is meant a dose
or a modulator or agent that produces therapeutic effects for which
it is administered. The exact dose will depend on the purpose of
the treatment, and will be ascertainable by one skilled in the art
using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage
Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of
Pharmaceutical Compounding (1999); Pickar, Dosage Calculations
(1999); and Remington: The Science and Practice of pharmacy, 20th
Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
In sensitized cells, the therapeutically effective dose can often
be lower than the conventional therapeutically effective dose for
non-sensitized cells.
[0118] The terms "overexpress," "overexpression" or "overexpressed"
interchangeably refer to a gene that is transcribed or translated
at a detectably greater level, usually in a cancer cell, in
comparison to a normal cell. Overexpression therefore refers to
both overexpression of protein and RNA (due to increased
transcription, post transcriptional processing, translation, post
translational processing, altered stability, and altered protein
degradation), as well as local overexpression due to altered
protein traffic patterns (increased nuclear localization), and
augmented functional activity, e.g., as a transcription factor, as
a DNA binding factor. Overexpression can be detected using
conventional techniques for detecting mRNA (i.e., RT-PCR, PCR,
hybridization) or proteins (i.e., ELISA, Western blots, flow
cytometry, immunofluorescence, immunohistochemical, DNA binding
assay techniques). Overexpression can be 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or more in comparison to a normal cell. In
certain instances, overexpression is 1-fold, 2-fold, 3-fold, 4-fold
or more higher levels of transcription or translation in comparison
to a normal cell.
[0119] The terms "underexpress," "underexpression" or
"underexpressed" interchangeably refer to a gene that is
transcribed or translated at a detectably lower level, usually in a
cancer cell, in comparison to a normal cell. Underexpression
therefore refers to both underexpression of protein and RNA (due to
decreased transcription, post transcriptional processing,
translation, post translational processing, altered stability, and
altered protein degradation), as well as local underexpression due
to altered protein traffic patterns (decreased nuclear
localization), and altered functional activity, e.g., as an enzyme.
Underexpression can be detected using conventional techniques for
detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins
(i.e., ELISA, Western blots, flow cytometry, immunofluorescence,
immunohistochemical, DNA binding assay techniques). Underexpression
can be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less in
comparison to a normal cell. In certain instances, underexpression
is 1-fold, 2-fold, 3-fold, 4-fold or more lower levels of
transcription or translation in comparison to a normal cell.
[0120] The terms "cancer-associated antigen" or "tumor-specific
marker" or "tumor marker" interchangeably refers to a molecule
(typically protein, carbohydrate or lipid) that is preferentially
expressed or over expressed in a cancer cell (e.g., on the cell
surface or intracellularly) in comparison to a normal cell, and
which is useful for the preferential targeting of a pharmacological
agent to the cancer cell. A marker or antigen can be expressed on
the cell surface or intracellularly. Oftentimes, a
cancer-associated antigen is a molecule that is overexpressed,
underexpressed in a cancer cell in comparison to a normal cell.
Oftentimes, a cancer-associated antigen is a molecule that is
inappropriately synthesized in the cancer cell, for instance, a
molecule that contains deletions, additions or mutations in
comparison to the molecule expressed on a normal cell. A cancer
associated antigen can have minimal degradation in the cancer cell.
Oftentimes, a cancer-associated antigen will be expressed
exclusively in a cancer cell and not synthesized or expressed in a
normal cell. Exemplified cell surface tumor markers include the
proteins c-erbB-2, and PSMA for prostate cancer. Exemplified
intracellular tumor markers include, for example, mutated tumor
suppressor or cell cycle proteins, including p53.
[0121] An "agonist" refers to an agent that binds to a polypeptide
or polynucleotide of the invention, stimulates, increases,
activates, facilitates, enhances activation, sensitizes or up
regulates the activity or expression of a polypeptide or
polynucleotide of the invention. An agonist may inhibit or activate
signaling pathways according to its action.
[0122] An "antagonist" refers to an agent that inhibits expression
of a polypeptide or polynucleotide of the invention or binds to,
partially or totally blocks stimulation, decreases, prevents,
delays activation, inactivates, desensitizes, or down regulates the
activity of a polypeptide or polynucleotide of the invention.
[0123] "Inhibitors," "activators," and "modulators" of expression
or of activity are used to refer to inhibitory, activating, or
modulating molecules, respectively, identified using in vitro and
in vivo assays for expression or activity, e.g., ligands, agonists,
antagonists, and their homologs and mimetics. They may act directly
or indirectly. The term "modulator" includes inhibitors and
activators. Inhibitors are agents that, e.g., inhibit expression,
e.g., translation, post-translational processing, stability,
degradation, or nuclear or cytoplasmic localization of a
polypeptide, or transcription, post transcriptional processing,
stability or degradation of a polynucleotide of the invention or
bind to, partially or totally block stimulation, DNA binding,
transcription factor activity or enzymatic activity, decrease,
prevent, delay activation, inactivate, desensitize, or down
regulate the activity of a polypeptide or polynucleotide of the
invention, e.g., antagonists. Activators are agents that, e.g.,
induce or activate the expression of a polypeptide or
polynucleotide of the invention or bind to, stimulate, increase,
open, activate, facilitate, enhance activation, DNA binding or
enzymatic activity, sensitize or up regulate the activity of a
polypeptide or polynucleotide of the invention, e.g., agonists.
Modulators include naturally occurring and synthetic ligands,
antagonists, agonists, small chemical molecules, antibodies,
inhibitory RNA molecules (i.e., siRNA or antisense RNA) and the
like. Assays to identify inhibitors and activators include, e.g.,
applying putative modulator compounds to cells, in the presence or
absence of a polypeptide or polynucleotide of the invention and
then determining the functional effects on a polypeptide or
polynucleotide of the invention activity. Samples or assays
comprising a polypeptide or polynucleotide of the invention that
are treated with a potential activator, inhibitor, or modulator are
compared to control samples without the inhibitor, activator, or
modulator to examine the extent of effect. Control samples
(untreated with modulators) are assigned a relative activity value
of 100%. Inhibition is achieved when the activity value of a
polypeptide or polynucleotide of the invention relative to the
control is about 80%, optionally 50% or 25-1 %. Activation is
achieved when the activity value of a polypeptide or polynucleotide
of the invention relative to the control is 110%, optionally 150%,
optionally 200-500%, or 1000-3000% higher.
[0124] The term "test compound" or "drug candidate" or "modulator"
or grammatical equivalents as used herein describes any molecule,
either naturally occurring or synthetic, e.g., protein,
oligopeptide (e.g., from about 5 to about 25 amino acids in length,
preferably from about 10 to 20 or 12 to 18 amino acids in length,
preferably 12, 15, or 18 amino acids in length), small organic
molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi,
oligonucleotide, etc. The test compound can be in the form of a
library of test compounds, such as a combinatorial or randomized
library that provides a sufficient range of diversity. Test
compounds are optionally linked to a fusion partner, e.g.,
targeting compounds, rescue compounds, dimerization compounds,
stabilizing compounds, addressable compounds, and other functional
moieties. Conventionally, new chemical entities with useful
properties are generated by identifying a test compound (called a
"lead compound") with some desirable property or activity, e.g.,
inhibiting activity, creating variants of the lead compound, and
evaluating the property and activity of those variant compounds.
Often, high throughput screening (HTS) methods are employed for
such an analysis.
[0125] A "small organic molecule" refers to an organic molecule,
either naturally occurring or synthetic, that has a molecular
weight of more than about 50 Daltons and less than about 2500
Daltons, preferably less than about 2000 Daltons, preferably
between about 100 to about 1000 Daltons, more preferably between
about 200 to about 500 Daltons.
[0126] The term "nitric oxide donor" or "NO donor" refers to any
compound capable of the intracellular delivery of nitric oxide.
Typically, an NO donor is any compound capable of denitrition that
releases nitric oxide. Also included are those compounds that can
be metabolized in vivo into a compound which delivers nitric oxide
(e.g., a prodrug form of a NO donor). An NO donor can be a
synthetic or naturally occurring organic chemical compound and can
be a polypeptide. Exemplified pharmaceutical agents that are NO
donors include arginine (L- and D-), amyl nitrite, isoamyl nitrite,
nitroglycerin, isosorbide dinitrate, isosorbide-5-mononitrate,
erythrityl tetranitrate. Nitric oxide synthases, both constitutive
and inducible forms, are also nitric oxide donors.
[0127] The term "inducer of inducible nitric oxide synthase (iNOS)"
or "activator of iNOS" refers to any compound that promotes the
expression (transcription or translation) and/or promotes that
catalytic activity of iNOS.
[0128] A "cell-cycle-specific" or "antimitotic" or
"cytoskeletal-interacting" drug interchangeably refer to any
pharmacological agent that blocks cells in mitosis. Generally,
cell-cycle-specific-drugs bind to the cytoskeletal protein tubulin
and block the ability of tubulin to polymerize into microtubules,
resulting in the arrest of cell division at metaphase. Exemplified
cell-cycle-specific drugs include vinca alkaloids, taxanes,
colchicine, and podophyllotoxin. Exemplified vinca alkaloids
include vinblastine, vincristine, vindesine and vinorelbine.
Exemplifed taxanes include paclitaxel and docetaxel. Another
example of a cytoskeletal-interacting drug includes
2-methoxyestradiol.
[0129] Rituximab refers to a chimeric murine/human monoclonal
antibody which targets the CD20 antigen which can be found on the
surface of normal and malignant B lymphocytes. Rituximab itself is
an IgG.sub.1 kappa immunoglobulin containing murine light- and
heavy-chain variable region sequences and human constant region
sequences. Rituximab is reported to have two heavy chains of 451
amino acids and two light chains of 213 amino acids (based on cDNA
analysis) with an approximate molecular weight of 145 kD. The
binding affinity of rituximab for the CD20 antigen of approximately
8.0 nM. While rituximab is referenced throughout the specification
other antibodies which target the CD20 antigen and trigger
signaling mediated by the CD20 antigen are also suitable.
[0130] An "siRNA" or "RNAi" refers to a nucleic acid that forms a
double stranded RNA, which double stranded RNA has the ability to
reduce or inhibit expression of a gene or target gene when the
siRNA expressed in the same cell as the gene or target gene.
"siRNA" or "RNAi" thus refers to the double stranded RNA formed by
the complementary strands. The complementary portions of the siRNA
that hybridize to form the double stranded molecule typically have
substantial or complete identity. In one embodiment, an siRNA
refers to a nucleic acid that has substantial or complete identity
to a target gene and forms a double stranded siRNA. Typically, the
siRNA is at least about 15-50 nucleotides in length (e.g., each
complementary sequence of the double stranded siRNA is 15-50
nucleotides in length, and the double stranded siRNA is about 15-50
base pairs in length, preferable about preferably about 20-30 base
nucleotides, preferably about 20-25 or about 24-29 nucleotides in
length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides in length.
[0131] "Determining the functional effect" refers to assaying for a
compound that increases or decreases a parameter that is indirectly
or directly under the influence of a polynucleotide or polypeptide
of the invention, e.g., measuring physical and chemical or
phenotypic effects. Such functional effects can be measured by any
means known to those skilled in the art, e.g., changes in
spectroscopic (e.g., fluorescence, absorbance, refractive index),
hydrodynamic (e.g., shape), chromatographic, or solubility
properties for the protein; measuring inducible markers or
transcriptional activation of the protein; measuring binding
activity or binding assays, e.g. binding to antibodies, binding to
DNA; measuring changes in ligand binding affinity; measurement of
calcium influx; measurement of the accumulation of an enzymatic
product of a polypeptide of the invention or depletion of an
substrate; changes in enzymatic activity, e.g., kinase activity,
measurement of changes in protein levels of a polypeptide of the
invention; measurement of RNA stability; G-protein binding; GPCR
phosphorylation or dephosphorylation; signal transduction, e.g.,
receptor-ligand interactions, second messenger concentrations
(e.g., cAMP, IP3, or intracellular Ca2+); identification of
downstream or reporter gene expression (CAT, luciferase,
.beta.-gal, GFP and the like), e.g., via chemiluminescence,
fluorescence, colorimetric reactions, antibody binding, inducible
markers, and ligand binding assays.
[0132] Samples or assays comprising a nucleic acid or protein
disclosed herein that are treated with a potential activator,
inhibitor, or modulator are compared to control samples without the
inhibitor, activator, or modulator to examine the extent of
inhibition. Control samples (untreated with inhibitors) are
assigned a relative protein activity value of 100%. Inhibition is
achieved when the activity value relative to the control is about
80%, preferably 50%, more preferably 25-0%. Activation is achieved
when the activity value relative to the control (untreated with
activators) is 110%, more preferably 150%, more preferably 200-500%
(i.e., two to five fold higher relative to the control), more
preferably 1000-3000% higher.
[0133] "Biological sample" or "tissue sample" includes sections of
tissues such as biopsy and autopsy samples, and frozen sections
taken for histologic purposes. Such samples include blood and blood
fractions or products (e.g., serum, plasma, platelets, red blood
cells, and the like), sputum, tissue, cultured cells, e.g., primary
cultures, explants, and transformed cells, stool, urine, etc. A
biological sample is typically obtained from a eukaryotic organism,
most preferably a mammal such as a primate e.g., chimpanzee or
human; cow; dog; cat; a rodent, e.g., guinea pig, rat, Mouse;
rabbit; or a bird; reptile; or fish.
[0134] A "biopsy" refers to the process of removing a tissue sample
for diagnostic or prognostic evaluation, and to the tissue specimen
itself. Any biopsy technique known in the art can be applied to the
diagnostic and prognostic methods of the present invention. The
biopsy technique applied will depend on the tissue type to be
evaluated (i.e., prostate, lymph node, liver, bone marrow, blood
cell), the size and type of the tumor (i.e., solid or suspended
(i.e., blood or ascites)), among other factors. Representative
biopsy techniques include excisional biopsy, incisional biopsy,
needle biopsy, surgical biopsy, and bone marrow biopsy. An
"excisional biopsy" refers to the removal of an entire tumor mass
with a small margin of normal tissue surrounding it. An "incisional
biopsy" refers to the removal of a wedge of tissue that includes a
cross-sectional diameter of the tumor. A diagnosis or prognosis
made by endoscopy or fluoroscopy can require a "core-needle biopsy"
of the tumor mass, or a "fine-needle aspiration biopsy" which
generally obtains a suspension of cells from within the tumor mass.
Biopsy techniques are discussed, for example, in Harrison 's
Principles of Internal Medicine, Kasper, et al., eds., 16th ed.,
2005, Chapter 70, and throughout Part V.
[0135] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated
region) as measured using a BLAST or BLAST 2.0 sequence comparison
algorithms with default parameters described below, or by manual
alignment and visual inspection (see, e.g., NCBI web site
http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are
then said to be "substantially identical." This definition also
refers to, or may be applied to, the compliment of a test sequence.
The definition also includes sequences that have deletions and/or
additions, as well as those that have substitutions. As described
below, the preferred algorithms can account for gaps and the like.
Preferably, identity exists over a region that is at least about 25
amino acids or nucleotides in length, or more preferably over a
region that is 50-100 amino acids or nucleotides in length.
[0136] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0137] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA, 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1987-2005, Wiley
Interscience)).
[0138] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al.,
J. Mol. Biol., 215:403-410 (1990), respectively. BLAST and BLAST
2.0 are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA, 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0139] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, and complements thereof. The term encompasses
nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally
occurring, and non-naturally occurring, which have similar binding
properties as the reference nucleic acid, and which are metabolized
in a manner similar to the reference nucleotides. Examples of such
analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0140] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.,
19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608
(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0141] A particular nucleic acid sequence also implicitly
encompasses "splice variants" and nucleic acid sequences encoding
truncated forms of functional or activated Bcl-2/Bcl-.sub.XL, AKT,
PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB, and transcription
factors AP-1 and STAT3. Similarly, a particular protein encoded by
a nucleic acid implicitly encompasses any protein encoded by a
splice variant or truncated form of that nucleic acid. "Splice
variants," as the name suggests, are products of alternative
splicing of a gene. After transcription, an initial nucleic acid
transcript may be spliced such that different (alternate) nucleic
acid splice products encode different polypeptides. Mechanisms for
the production of splice variants vary, but include alternate
splicing of exons. Alternate polypeptides derived from the same
nucleic acid by read-through transcription are also encompassed by
this definition. Any products of a splicing reaction, including
recombinant forms of the splice products, are included in this
definition. Nucleic acids can be truncated at the 5' end or at the
3' end. Polypeptides can be truncated at the N-terminal end or the
C-terminal end. Truncated versions of nucleic acid or polypeptide
sequences can be naturally occurring or recombinantly created.
Truncated forms of YY1 are described, for example, in Begon et al.,
J Biol Chem, 280:24428 (2005); Krippner-Heidenreich, et al., Mol
Cell Biol, 25:3704 (2005); Nishiyama et al., Biosci Biotechnol
Biochem, 67:654 (2003); and Berndt et al., J Neurochem, 77:935
(2001).
[0142] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0143] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0144] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0145] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0146] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0147] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0148] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include .sup.32p, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins which can be made detectable,
e.g., by incorporating a radiolabel into the peptide or used to
detect antibodies specifically reactive with the peptide.
[0149] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0150] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0151] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0152] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al., supra.
[0153] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures may
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.degree. C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. C. for 1-2
min. Protocols and guidelines for low and high stringency
amplification reactions are provided, e.g., in Innis et al., PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y. (1990)).
[0154] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0155] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0156] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature, 348:552-554 (1990)).
[0157] For preparation of antibodies, e.g., recombinant,
monoclonal, or polyclonal antibodies, many technique known in the
art can be used (see, e.g., Kohler & Milstein, Nature,
256:495-497 (1975); Kozbor et al., Immunology Today, 4:72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988);
and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986)). The genes encoding the heavy and light chains of an
antibody of interest can be cloned from a cell, e.g., the genes
encoding a monoclonal antibody can be cloned from a hybridoma and
used to produce a recombinant monoclonal antibody. Gene libraries
encoding heavy and light chains of monoclonal antibodies can also
be made from hybridoma or plasma cells. Random combinations of the
heavy and light chain gene products generate a large pool of
antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3.sup.rd ed. 1997)). Techniques for the production of
single chain antibodies or recombinant antibodies (U.S. Pat. No.
4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized or human antibodies (see, e.g., U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
Marks et al., Bio/Technology, 10:779-783 (1992); Lonberg et al.,
Nature, 368:856-859 (1994); Morrison, Nature, 368:812-13 (1994);
Fishwild et al., Nature Biotechnology, 14:845-51 (1996); Neuberger,
Nature Biotechnology, 14:826 (1996); and Lonberg & Huszar,
Intern. Rev. Immunol., 13:65-93 (1995)). Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al., Nature, 348:552-554 (1990);
Marks et al., Biotechnology, 10:779-783 (1992)). Antibodies can
also be made bispecific, i.e., able to recognize two different
antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J.,
10:3655-3659 (1991); and Suresh et al., Methods in Enzymology,
121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two
covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat.
No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
[0158] Methods for humanizing or primatizing non-human antibodies
are well known in the art. Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. These non-human amino acid residues are often
referred to as import residues, which are typically taken from an
import variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (see, e.g., Jones et
al., Nature, 321:522-525 (1986); Riechmann et al., Nature,
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)
and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such humanized
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0159] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0160] In one embodiment, the antibody is conjugated to an
"effector" moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a therapeutic moiety. In one
aspect the antibody modulates the activity of the protein.
[0161] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies can be
selected to obtain only those polyclonal antibodies that are
specifically immunoreactive with the selected antigen and not with
other proteins. This selection may be achieved by subtracting out
antibodies that cross-react with other molecules. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988) for a description of
immumunoassay formats and conditions that can be used to determine
specific immunoreactivity).
[0162] By "therapeutically effective amount or dose" or "sufficient
amount or dose" herein is meant a dose that produces effects for
which it is administered. The exact dose will depend on the purpose
of the treatment, and will be ascertainable by one skilled in the
art using known techniques (see, e.g., Lieberman, Pharmaceutical
Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and
Technology of Pharmaceutical Compounding (1999); Pickar, Dosage
Calculations (1999); and Remington: The Science and Practice of
Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams
& Wilkins).
[0163] The term "pharmaceutically acceptable salts" or
"pharmaceutically acceptable carrier" is meant to include salts of
the active compounds which are prepared with relatively nontoxic
acids or bases, depending on the particular substituents found on
the compounds described herein. When compounds of the present
invention contain relatively acidic functionalities, base addition
salts can be obtained by contacting the neutral form of such
compounds with a sufficient amount of the desired base, either neat
or in a suitable inert solvent. Examples of pharmaceutically
acceptable base addition salts include sodium, potassium, calcium,
ammonium, organic amino, or magnesium salt, or a similar salt. When
compounds of the present invention contain relatively basic
functionalities, acid addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired acid, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, e.g.,
Berge et al., Journal of Pharmaceutical Science, 66:1-19 (1977)).
Certain specific compounds of the present invention contain both
basic and acidic functionalities that allow the compounds to be
converted into either base or acid addition salts. Other
pharmaceutically acceptable carriers known to those of skill in the
art are suitable for the present invention.
[0164] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents, but otherwise the
salts are equivalent to the parent form of the compound for the
purposes of the present invention.
[0165] In addition to salt forms, the present invention provides
compounds which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0166] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are intended to be encompassed within the scope of the
present invention. Certain compounds of the present invention may
exist in multiple crystalline or amorphous forms. In general, all
physical forms are equivalent for the uses contemplated by the
present invention and are intended to be within the scope of the
present invention.
[0167] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are all intended to be encompassed within the scope of the present
invention.
Assays for Modulators of Cell Gene Products
[0168] Modulation of functional or activated gene products (e.g.,
Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB,
and transcription factors AP-1 and STAT3), and corresponding
modulation of cellular, e.g., tumor cell, proliferation, can be
assessed using a variety of in vitro and in vivo assays, including
cell-based models. Such assays can be used to test for cell gene
products or inhibitors and activators of functional or activated
Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB,
and transcription factors AP-1 and STAT3 transcription or
translation, or protein activity, and consequently, inhibitors and
activators of cellular proliferation, including modulators of
chemotherapeutic and immunotherapeutic sensitivity and toxicity.
Assays for modulation include cell-viability, cell proliferation,
cell responses to apoptotic stimuli, gene transcription, mRNA
arrays, kinase or phosphatase activity, interaction with other
proteins including other transcription factors, and DNA binding,
gene transfection assays, siRNA, Western assays, reporters assays,
and intracellular localization by confocal microscopy. Such
modulators are useful for treating disorders related to
pathological cell proliferation, e.g., cancer, autoimmunity, aging.
Modulators can be tested using in vivo well cells expressing
functional or activated Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY 1,
NF.kappa.B, NIK, IKK, IKB, and transcription factors AP-1 and STAT3
and in vitro well, either recombinant or naturally occurring YY1
protein, preferably human YY1. Wild type as well as truncated and
alternatively spliced forms of functional or activated
Bcl-2/Bcl-.sub.XL, AKT, PTEN, Fas, YY1, NF.kappa.B, NIK, IKK, IKB,
and transcription factors AP-1 and STAT3 are useful targets.
[0169] Measurement of cellular proliferation by modulation with a
protein or a nucleic acid, either recombinant or naturally
occurring, can be performed using a variety of assays, in vitro, in
vivo, and ex vivo, as described herein. A suitable physical,
chemical or phenotypic change that affects activity, e.g.,
enzymatic activity such as kinase activity, cell proliferation, or
ligand binding (e.g., a protein or nucleic acid receptor) can be
used to assess the influence of a test compound on the polypeptide
of this invention. When the functional effects are determined using
intact cells or animals, one can also measure a variety of effects,
such as, ligand binding, DNA binding, kinase activity,
transcriptional changes to both known and uncharacterized genetic
markers (e.g., Northern blots), changes in cell metabolism, changes
related to cellular proliferation, cell surface marker expression,
DNA synthesis, marker and dye dilution assays (e.g., GFP and cell
tracker assays), contact inhibition, tumor growth in nude mice,
reporter assays, overexpression or knockout gene products, and
siRNA, etc.
[0170] In Vitro Assays
[0171] Assays to identify modulator sor compounds with modulating
activity can be performed in vitro. Such assays can use a full
length protein or a variant thereof, or a mutant thereof, a
truncated form or a fragment of a protein. Purified recombinant or
naturally occurring protein can be used in the in vitro methods of
the invention. In addition to purified protein, the recombinant or
naturally occurring protein can be part of a cellular lysate or a
cell membrane. As described below, the binding assay can be either
solid state or soluble. Preferably, the protein or membrane is
bound to a solid support, either covalently or non-covalently.
Often, the in vitro assays of the invention are substrate or ligand
binding or affinity assays, either non-competitive or competitive.
Other in vitro assays include measuring changes in spectroscopic
(e.g., fluorescence, absorbance, refractive index), hydrodynamic
(e.g., shape), chromatographic, or solubility properties for the
protein. Other in vitro assays include enzymatic activity assays,
such as phosphorylation or autophosphorylation assays). Preferred
in vitro assay systems include DNA binding assays (EMSA). In
addition, cell based reporter assays, whole cell treatment assays
with checking for a gene product by Western, PCR, and microarray
methods for example can be used. Such products can be analyzed by
immunohistochemistry, immunofluorescence, confocal and microscopy,
for example.
[0172] In one embodiment, a high throughput binding assay is
performed in which the protein, a truncated form or a fragment
thereof is contacted with a potential modulator and incubated for a
suitable amount of time. In one embodiment, the potential modulator
is bound to a solid support, and the protein is added. In another
embodiment, the protein is bound to a solid support. A wide variety
of modulators can be used, as described herein, including small
organic molecules, peptides, antibodies, and binding protein or
nucleic acid analogs. A wide variety of assays can be used to
identify modulator binding, including labeled protein-protein
binding assays, electrophoretic mobility shifts, immunoassays,
enzymatic assays such as kinase assays, and the like. In some
cases, the binding of the candidate modulator is determined through
the use of competitive binding assays, where interference with
binding of a known ligand or substrate is measured in the presence
of a potential modulator.
[0173] In one embodiment, microtiter plates are first coated with
either a protein or a binding protein (ie. antibody, transcription
factors, etc.) or nucleic acid, and then exposed to one or more
test compounds potentially capable of inhibiting the binding of a
protein to a binding protein or nucleic acid. A labeled (i.e.,
fluorescent, enzymatic, radioactive isotope) binding partner of the
coated protein, either a binding protein or nucleic acid, or a
protein, is then exposed to the coated protein and test compounds.
Unbound protein (or nucleic acid) is washed away as necessary in
between exposures to a protein, a binding protein or nucleic acid,
or a test compound. An absence of detectable signal indicates that
the test compound inhibited the binding interaction between a
protein and a binding protein or nucleic acid. The presence of
detectable signal (i.e., fluorescence, calorimetric, radioactivity)
indicates that the test compound did not inhibit the binding
interaction between a protein and a binding protein or nucleic
acid. One can also use chromatographic techniques, for example
HPLC, and evaluate elution profiles of protein alone and protein
complexed with other factors, including DNA and/or other
transcription factors. The presence or absence of detectable signal
is compared to a control sample that was not exposed to a test
compound, which exhibits uninhibited signal. In some embodiments
the binding partner is unlabeled, but exposed to a labeled antibody
that specifically binds the binding partner.
[0174] Cell-based in Vivo Assays
[0175] In another embodiment, protein is expressed in a cell, and
functional, e.g., physical and chemical or phenotypic, changes are
assayed to identify modulators of cellular proliferation, e.g.,
tumor cell proliferation. In some embodiments, as exemplified in
some instances herein, the cells can be chemo- or
treatment-resistant cell lines or clones, including clones of
lymphoma, or leukemia cells. Cells expressing recombinant or
endogenous proteins can also be used in binding assays and
enzymatic assays. Preferably, the cells overexpress or under
express protein in comparison to a normal cell of the same type.
Any suitable functional effect can be measured, as described
herein. For example, cellular morphology (e.g., cell volume,
nuclear volume, cell perimeter, and nuclear perimeter), ligand
binding, kinase activity, apoptosis, cell surface marker
expression, cellular proliferation, cellular localization of
proteins or transcripts, DNA binding, GFP positivity and dye
dilution assays (e.g., cell tracker assays with dyes that bind to
cell membranes), DNA synthesis assays (e.g., .sup.3H-thymidine and
fluorescent DNA-binding dyes such as BrdU or Hoechst dye with FACS
analysis), are all suitable assays to identify potential modulators
using a cell based system. Suitable cells for such cell based
assays include both primary cancer or tumor cells and cell lines,
as described herein, e.g., A549 (lung), MCF7 (breast, p53
wild-type), H1299 (lung, p53 null), Hela (cervical), PC3 (prostate,
p53 mutant), MDA-MB-231 (breast, p53 wild-type). Variants derived
from these cell lines with specific gene modification will also be
used. Cancer cell lines can be p53 mutant, p53 null, or express
wild type p53. The protein can be naturally occurring or
recombinant. Also, truncated forms or fragments or chimeric
proteins can be used in cell based assays.
[0176] Cellular polypeptide levels can be determined by measuring
the level of protein or mRNA. The level of protein or related
proteins are measured using immunoassays such as western blotting,
ELISA, immunofluorescence and the like with an antibody that
selectively binds to the polypeptide or a fragment thereof. For
measurement of mRNA, amplification, e.g., using PCR, RT-PCR, LCR,
or hybridization assays, e.g., northern hybridization, RNAse
protection, dot blotting, are preferred. The level of protein or
mRNA is detected using directly or indirectly labeled detection
agents, e.g., fluorescently or radioactively labeled nucleic acids,
radioactively or enzymatically labeled antibodies, and the like, as
described herein. It is also useful to observe protein
translocation into the nucleus and other cellular compartments by,
for example, confocal microscopy.
[0177] Alternatively, protein expression can be measured using a
reporter gene system. Such a system can be devised using a protein
promoter which modulates transcription of the protein, or a protein
responsive site, which is modulated by binding of the protein,
operably linked to a reporter gene, including chloramphenicol
acetyltransferase, firefly luciferase, bacterial luciferase,
.beta.-galactosidase, green fluorescent protein (GFP) and alkaline
phosphatase. Furthermore, the protein of interest can be used as an
indirect reporter via attachment to a second reporter such as red
or green fluorescent protein (see, e.g., Mistili & Spector,
Nature Biotechnology, 15:961-964 (1997)). The reporter construct is
typically transfected into a cell. After treatment with a potential
modulator, the amount of reporter gene transcription, translation,
or activity is measured according to standard techniques known to
those of skill in the art. In a preferred embodiment, plasmids that
allow for stable transfection are used.
[0178] Animal Models
[0179] Animal models of cellular proliferation also find use in
screening for modulators of cellular proliferation. Similarly,
transgenic animal technology including gene knockout technology,
for example, as a result of homologous recombination with an
appropriate gene targeting vector, or gene overexpression, will
result in the absence or increased expression of the protein. The
same technology can also be applied to make knock-out cells. When
desired, tissue-specific expression or knockout of the protein may
be necessary. Transgenic animals generated by such methods find use
as animal models of cellular proliferation and are additionally
useful in screening for modulators of cellular proliferation.
[0180] Knock-out cells and transgenic mice can be made by insertion
of a marker gene or other heterologous gene into an endogenous gene
site in the mouse genome via homologous recombination. Such mice
can also be made by substituting an endogenous gene with a mutated
version of the gene, or by mutating an endogenous gene, e.g., by
exposure to carcinogens.
[0181] In particular, human tumor xenografts can be used and the
tissues be examined for the effects of a treatment on gene
modifications by IHC, ISH, RT-PCR, Western, etc. Treatments can
also be with various sensitizing agents in combination with
drugs.
[0182] A DNA construct is introduced into the nuclei of embryonic
stem cells. Cells containing the newly engineered genetic lesion
are injected into a host mouse embryo, which is re-implanted into a
recipient female. Some of these embryos develop into chimeric mice
that possess germ cells partially derived from the mutant cell
line. Therefore, by breeding the chimeric mice it is possible to
obtain a new line of mice containing the introduced genetic lesion
(see, e.g., Capecchi et al., Science, 244:1288 (1989)). Chimeric
targeted mice can be derived according to Hogan et al.,
Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring
Harbor Laboratory (1988), Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,
D.C., (1987), and Pinkert, Transgenic Animal Technology: A
Laboratory Handbook, Academic Press (2003).
[0183] Exemplary Assays
[0184] Soft Agar Growth or Colony Formation in Suspension
[0185] Normal cells require a solid substrate to attach and grow.
When the cells are transformed, they lose this phenotype and grow
detached from the substrate. For example, transformed cells can
grow in stirred suspension culture or suspended in semi-solid
media, such as semi-solid or soft agar. The transformed cells, when
transfected with tumor suppressor genes, regenerate normal
phenotype and require a solid substrate to attach and grow.
[0186] Soft agar growth or colony formation in suspension assays
can be used to identify modulators. Typically, transformed host
cells (e.g., cells that grow on soft agar) are used in this assay.
For example, RKO or HCT116 cell lines can be used. Techniques for
soft agar growth or colony formation in suspension assays are
described in Freshney, Culture of Animal Cells a Manual of Basic
Technique, 3.sup.rd ed., Wiley-Liss, New York (1994), herein
incorporated by reference. See also, the methods section of
Garkavtsev et al. (1996), supra, herein incorporated by
reference.
[0187] Contact Inhibition and Density Limitation of Growth
[0188] Normal cells typically grow in a flat and organized pattern
in a petri dish until they touch other cells. When the cells touch
one another, they are contact inhibited and stop growing. When
cells are transformed, however, the cells are not contact inhibited
and continue to grow to high densities in disorganized foci. Thus,
the transformed cells grow to a higher saturation density than
normal cells. This can be detected morphologically by the formation
of a disoriented monolayer of cells or rounded cells in foci within
the regular pattern of normal surrounding cells. Alternatively,
labeling index with [.sup.3H]-thymidine at saturation density can
be used to measure density limitation of growth. See Freshney
(1994), supra. The transformed cells, when contacted with cellular
proliferation modulators, regenerate a normal phenotype and become
contact inhibited and would grow to a lower density.
[0189] Contact inhibition and density limitation of growth assays
can be used to identify modulators which are capable of inhibiting
abnormal proliferation and transformation in host cells. Typically,
transformed host cells (e.g., cells that are not contact inhibited)
are used in this assay. For example, RKO or HCT116 cell lines can
be used. In this assay, labeling index with [.sup.3H]-thymidine at
saturation density is a preferred method of measuring density
limitation of growth. Transformed host cells are contacted with a
potential modulator and are grown for 24 hours at saturation
density in non-limiting medium conditions. The percentage of cells
labeling with [.sup.3H]-thymidine is determined
autoradiographically. See, Freshney (1994), supra. The host cells
contacted with a modulator would give arise to a lower labeling
index compared to control (e.g., transformed host cells transfected
with a vector lacking an insert).
[0190] Growth Factor or Serum Dependence
[0191] Growth factor or serum dependence can be used as an assay to
identify modulators. Transformed cells have a lower serum
dependence than their normal counterparts (see, e.g., Temin, J.
Natl. Cancer Insti., 37:167-175 (1966); Eagle et al., J. Exp. Med.,
131:836-879 (1970)); Freshney, supra. This is in part due to
release of various growth factors by the transformed cells. When
transformed cells are contacted with a modulator, the cells would
reacquire serum dependence and would release growth factors at a
lower level.
[0192] Tumor Specific Markers Levels
[0193] Tumor cells release an increased amount of certain factors
(hereinafter "tumor specific markers") than their normal
counterparts. For example, plasminogen activator (PA) is released
from human glioma at a higher level than from normal brain cells
(see, e.g., Gullino, Angiogenesis, tumor vascularization, and
potential interference with tumor growth. In Mihich (ed.):
"Biological Responses in Cancer." New York, Academic Press, pp.
178-184 (1985)). Similarly, tumor angiogenesis factor (TAF) is
released at a higher level in tumor cells than their normal
counterparts. See, e.g., Folkman, Angiogenesis and cancer, Sem
Cancer Biol. (1992)). Other exemplified tumor specific markers
include growth factors and cytokines.
[0194] Tumor specific markers can be assayed to identify modulators
which decrease the level of release of these markers from host
cells. Typically, transformed or tumorigenic host cells are used.
Various techniques which measure the release of these factors are
described in Freshney (1994), supra. Also, see, Unkless et al., J.
Biol. Chem., 249:4295-4305 (1974); Strickland & Beers, J. Biol.
Chem., 251:5694-5702 (1976); Whur et al., Br. J. Cancer, 42:305-312
(1980); Gulino, Angiogenesis, tumor vascularization, and potential
interference with tumor growth. In Mihich, E. (ed): "Biological
Responses in Cancer." New York, Plenum (1985); Freshney, Anticancer
Res., 5:111-130 (1985).
[0195] Invasiveness into Matrigel
[0196] The degree of invasiveness into Matrigel or some other
extracellular matrix constituent can be used as an assay to
identify modulators which are capable of inhibiting abnormal cell
proliferation and tumor growth. Tumor cells exhibit a good
correlation between malignancy and invasiveness of cells into
Matrigel or some other extracellular matrix constituent. In this
assay, tumorigenic cells are typically used as host cells.
Therefore, modulators can be identified by measuring changes in the
level of invasiveness between the host cells before and after the
introduction of potential modulators. If a compound modulates a
protein, its expression in tumorigenic host cells would affect
invasiveness.
[0197] Techniques described in Freshney (1994), supra, can be used.
Briefly, the level of invasion of host cells can be measured by
using filters coated with Matrigel or some other extracellular
matrix constituent. Penetration into the gel, or through to the
distal side of the filter, is rated as invasiveness, and rated
histologically by number of cells and distance moved, or by
prelabeling the cells with .sup.125I and counting the radioactivity
on the distal side of the filter or bottom of the dish. See, e.g.,
Freshney (1984), supra.
[0198] G.sub.0/G.sub.1 Cell Cycle Arrest Analysis
[0199] G.sub.0/G.sub.1 cell cycle arrest can be used as an assay to
identify modulators. In this assay, cell lines, such as RKO or
HCT116, can be used to screen modulators. The cells can be
co-transfected with a construct comprising a marker gene, such as a
gene that encodes green fluorescent protein, or a cell tracker dye.
Methods known in the art can be used to measure the degree of
G.sub.1 cell cycle arrest. For example, a propidium iodide signal
can be used as a measure for DNA content to determine cell cycle
profiles on a flow cytometer. The percent of the cells in each cell
cycle can be calculated. Cells contacted with a modulator would
exhibit, e.g., a higher number of cells that are arrested in
G.sub.0/G.sub.1 phase compared to control.
[0200] Apoptotic Pathways and Cell Signaling
[0201] Additionally, can be analyzed as, for instance, exemplified
herein. Screening can done using microarrays.
[0202] Tumor Growth in Vivo
[0203] Effects of modulators on cell growth can be tested in
transgenic or immune-suppressed mice. Knock-out transgenic mice can
be made, in which the endogenous gene is disrupted. Such knock-out
mice can be used to study effects of the gene and its protein,
e.g., as a cancer model, as a means of assaying in vivo for
compounds that modulate the protein, and to test the effects of
restoring a wild-type or mutant gene to a knock-out mouse.
[0204] Knock-out cells and transgenic mice can be made by insertion
of a marker gene or other heterologous gene into the endogenous
gene site in the mouse genome via homologous recombination. Such
mice can also be made by substituting the endogenous gene with a
mutated version of the gene, or by mutating the endogenous gene,
e.g., by exposure to carcinogens.
[0205] A DNA construct is introduced into the nuclei of embryonic
stem cells. Cells containing the newly engineered genetic lesion
are injected into a host mouse embryo, which is re-implanted into a
recipient female. Some of these embryos develop into chimeric mice
that possess germ cells partially derived from the mutant cell
line. Therefore, by breeding the chimeric mice it is possible to
obtain a new line of mice containing the introduced genetic lesion
(see, e.g., Capecchi et al., Science, 244:1288 (1989)). Chimeric
targeted mice can be derived according to Hogan et al.,
Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring
Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,
D.C., (1987). These knock-out mice can be used as hosts to test the
effects of various modulators on cell growth.
[0206] Alternatively, various immune-suppressed or immune-deficient
host animals can be used. For example, genetically athymic "nude"
mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst., 52:921
(1974)), a SCID mouse, a thymectomized mouse, or an irradiated
mouse (see, e.g., Bradley et al., Br. J. Cancer, 38:263 (1978);
Selby et al., Br. J. Cancer, 41:52 (1980)) can be used as a host.
Transplantable tumor cells (typically about 10.sup.6 cells)
injected into isogenic hosts will produce invasive tumors in a high
proportions of cases, while normal cells of similar origin will
not. Hosts are treated with modulators, e.g., by injection,
optionally in combination with other cancer therapeutic agents,
including chemotherapy, radiotherapy, immunotherapy or hormonal
therapy. After a suitable length of time, preferably 4-8 weeks,
tumor growth is measured (e.g., by volume or by its two largest
dimensions) and compared to the control. Tumors that have
statistically significant reduction (using, e.g., Student's T test)
are said to have inhibited growth. Using reduction of tumor size as
an assay, modulators which are capable, e.g., of inhibiting
abnormal cell proliferation or sensitizing tumor cells to cancer
therapies, can be identified.
[0207] In immune-suppressed or immune-deficient host animals, the
inoculating tumor cells preferably overexpress or underexpress the
gene or protein of interest. The inoculating tumor cells are also
preferably resistant to conventionally used cancer therapies.
Exemplified modulators include rituxamib, siRNA, NO donors,
NF-.kappa.B inhibitors (i.e., dehydroxymethylepoxyquinomicin
(DHMEQ)), proteasome inhibitors (i.e., Bortezomib, Velcade), and
microtubule inhibitors (i.e., 2-Methoxyestradiol (2ME2) and
vincristine). In one example, tumor cells resistant to death
receptor-induced (e.g., DR5) apoptosis are inoculated as xenografts
in SCID mice. The mice are subsequently treated with one or more
inhibitors (siRNA, NO donors, NF-.kappa.B inhibitors, etc.)
combined with a death receptor agonist (e.g., a monoclonal antibody
to DR5 or TRAIL).
[0208] Murine, rodent and other animal tumor models for studying
cancer are generally described, for example, in Immunodeficient
Animals: Models for Cancer Research, Arnold et al., eds., 1996, S
Karger Pub; Tumor Models in Cancer Research, Teicher, ed., 2002,
Human Press; and Mouse Models of Cancer, Holland, ed., 2004, John
Wiley & Sons. Specific murine tumor models for several
different cancers have been described, including for example,
metastatic colon cancer (Luo, et al., Cancer Cell, 6:297 (2004)),
breast cancer (Rahman & Sarkar, Cancer Res, 65:364 (2005)),
cholangiocarcinoma (Chen et al., World J Gastroenterol, 11:726
(2005)), and prostate cancer (Tsingotjidou et al., Anticancer Res,
21:971 (2001) and U.S. Pat. No. 6,107,540).
Screening Methods
[0209] The present invention also provides methods of identifying
compounds that inhibit cancer growth or progression, for example,
by inhibiting the binding of a protein to a binding protein or a
nucleic acid. The compounds find use in inhibiting the growth of
and promoting the regression of a tumor that has altered expression
of a protein, for example, prostate cancer, ovarian cancer, lung
cancer, renal cancer, breast cancer, colon cancer, leukemias,
B-cell lymphomas (e.g., non-Hodgkin's lymphomas, including
Burkitt's, Small Cell, and Large Cell lymphomas), hepatocarcinoma
or multiple myeloma. The identified compounds can inhibit cancer
growth or progression alone, or when used in combination with other
cancer therapies, including chemotherapies, radiation therapies,
hormonal therapies and immunotherapies.
[0210] Using the assays described herein, one can identify lead
compounds that are suitable for further testing to identify those
that are therapeutically effective modulating agents. One
particularly useful assay system utilized a reporter system where a
reporter gene (i.e., luciferase or GFP) is operably linked to a
promoter sequence comprising a binding sequence. Compounds of
interest can be either synthetic or naturally occurring.
[0211] Screening assays can be carried out in vitro or in vivo.
Typically, initial screening assays are carried out in vitro, and
can be confirmed in vivo using cell based assays or animal models.
For instance, proteins of the regenerating gene family are involved
with cell proliferation. Therefore, compounds that inhibit a
protein or nucleic acid can inhibit cell proliferation in
comparison to cells unexposed to a test compound. Also, the protein
of interest can be involved with tissue injury responses,
inflammation, and dysplasia. In animal models, compounds that
inhibit the protein can, for example, inhibit wound healing or the
progression of dysplasia in comparison to an animal unexposed to a
test compound. See,for example, Zhang et al., World J Gastroenter,
9:2635-41 (2003).
[0212] Usually, a compound that inhibits the protein is synthetic,
but it can also be naturally occurring. The screening methods are
designed to screen large chemical or polymer (i.e., inhibitory RNA,
including siRNA and antisense RNA, peptides, small organic
molecules, etc.) libraries by automating the assay steps and
providing compounds from any convenient source to the assays, which
are typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays).
[0213] The invention provides in vitro assays for modulating a
protein or nucleic acid in a high throughput format. For each of
the assay formats described, "no modulator" control reactions which
do not include a modulator provide a background level interaction.
In the high throughput assays of the invention, it is possible to
screen up to several thousand different modulators in a single day.
In particular, each well of a microtiter plate can be used to run a
separate assay against a selected potential modulator, or, if
concentration or incubation time effects are to be observed, every
5-10 wells can test a single modulator. Thus, a single standard
microtiter plate can assay about 100 (96) modulators. If 1536 well
plates are used, then a single plate can easily assay from about
100- about 1500 different compounds. It is possible to assay many
different plates per day; assay screens for up to about
6,000-20,000, and even up to about 100,000-1,000,000 different
compounds is possible using the integrated systems of the
invention. The steps of labeling, addition of reagents, fluid
changes, and detection are compatible with full automation, for
instance using programmable robotic systems or "integrated systems"
commercially available, for example, through BioTX Automation,
Conroe, Tex.; Qiagen, Valencia, Calif.; Beckman Coulter, Fullerton,
Calif.; and Caliper Life Sciences, Hopkinton, Mass.
[0214] Essentially, any chemical compound can be tested as a
potential inhibitor of protein or nucleic acid for use in the
methods of the invention. Most preferred are generally compounds
that can be dissolved in aqueous or organic (especially DMSO-based)
solutions are used. It will be appreciated that there are many
suppliers of chemical compounds, including Sigma (St. Louis, Mo.),
Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka
Chemika-Biochemica Analytika (Buchs Switzerland), as well as
providers of small organic molecule and peptide libraries ready for
screening, including Chembridge Corp. (San Diego, Calif.),
Discovery Partners International (San Diego, Calif.), Triad
Therapeutics (San Diego, Calif.), Nanosyn (Menlo Park, Calif.),
Affymax (Palo Alto, Calif.), ComGenex (South San Francisco,
Calif.), and Tripos, Inc. (St. Louis, Mo.).
[0215] Compounds also include those that can regulate transcription
and post-transcriptional processing and compounds that can regulate
gene expression under the control of a gene or protein of interest.
Reporter systems can be used for this analysis.
[0216] In one preferred embodiment, inhibitors of protein or
nucleic acid are identified by screening a combinatorial library
containing a large number of potential therapeutic compounds
(potential modulator compounds). Such "combinatorial chemical or
peptide libraries" can be screened in one or more assays, as
described herein, to identify those library members (particular
chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0217] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0218] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art (see, for
example, Beeler et al., Curr Opin Chem Biol., 9:277 (2005) and
Shang and Tan, Curr Opin Chem Biol., 9:248 (2005). Libraries of use
in the present invention can be composed of amino acid compounds,
nucleic acid compounds, carbohydrates or small organic compounds.
Carbohydrate libraries have been described in, for example, Liang
et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.
5,593,853.
[0219] Representative amino acid compound libraries include, but
are not limited to, peptide libraries (see, e.g., U.S. Pat. Nos.
5,010,175; 6,828,422 and 6,844,161; Furka, Int. J. Pept. Prot.
Res., 37:487-493 (1991); Houghton et al., Nature, 354:84-88 (1991)
and Eichler, Comb Chem High Throughput Screen., 8:135 (2005)),
peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT
Publication WO 93/20242), random bio-oligomers (PCT Publication No.
WO 92/00091), vinylogous polypeptides (Hagihara et al., J. Amer.
Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimetics with
.beta.-D-glucose scaffolding (Hirschmann et al., J. Amer. Chem.
Soc., 114:9217-9218 (1992)), peptide nucleic acid libraries (see,
e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., U.S.
Pat. Nos. 6,635,424 and 6,555,310; PCT/US96/10287, and Vaughn et
al., Nature Biotechnology, 14(3):309-314 (1996)), and peptidyl
phosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)).
[0220] Representative nucleic acid compound libraries include, but
are not limited to, genomic DNA, cDNA, mRNA, inhibitory RNA (RNAi,
siRNA) and antisense RNA libraries. See, Ausubel, Current Protocols
in Molecular Biology, supra, and Sambrook and Russell, Molecular
Cloning: A Laboratory Manual, 2000, Cold Spring Harbor Laboratory
Press. Nucleic acid libraries are described in, for example, U.S.
Pat. Nos. 6,706,477; 6,582,914; and 6,573,098. cDNA libraries are
described in, for example, U.S. Pat. Nos. 6,846,655; 6,841,347;
6,828,098; 6,808,906; 6,623,965; and 6,509,175. RNA libraries, for
example, ribozyme, RNA interference or siRNA libraries, are
reviewed in, for example, Downward, Cell, 121:813 (2005) and Akashi
et al., Nat Rev Mol Cell Biol., 6:413 (2005). Antisense RNA
libraries are described in, for example, U.S. Pat. Nos. 6,586,180
and 6,518,017.
[0221] Representative small organic molecule libraries include, but
are not limited to, diversomers such as hydantoins, benzodiazepines
and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA,
90:6909-6913 (1993)), analogous organic syntheses of small compound
libraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),
oligocarbamates (Cho et al., Science 261:1303 (1993));
benzodiazepines (U.S. Pat. No. 5,288,514; and Baum, C&EN,
January 18, page 33 (1993)); isoprenoids (e.g., U.S. Pat. No.
5,569,588); thiazolidinones and metathiazanones (e.g., U.S. Pat.
5,549,974); pyrrolidines (e.g., U.S. Pat. Nos. 5,525,735 and
5,519,134); morpholino compounds (e.g., U.S. Pat. No. 5,506,337);
tetracyclic benzimidazoles (e.g., U.S. Pat. No. 6,515,122);
dihydrobenzpyrans (e.g., U.S. Pat. No. 6,790,965); amines (e.g.,
U.S. Pat. No. 6,750,344); phenyl compounds (e.g., U.S. Pat. No.
6,740,712); azoles, (e.g., U.S. Pat. No. 6,683,191); pyridine
carboxamides or sulfonamides (e.g., U.S. Pat. No. 6,677,452);
2-aminobenzoxazoles (e.g., U.S. Pat. No. 6,660,858); isoindoles,
isooxyindoles, or isooxyquinolines (e.g., U.S. Pat. No. 6,667,406);
oxazolidinones (e.g., U.S. Pat. No. 6,562,844); and hydroxylamines
(e.g., U.S. Pat. No. 6,541,276).
[0222] Of particular interest are libraries of nitric oxide donor
compounds, for example, libraries of molecules with core structures
like the nitric oxide donor compounds disclosed in U.S. Pat. Nos.
6,897,218; 6,897,194; 6,780,849; 6,642,260; 6,538,033; 6,451,337;
and 5,698,738 (see also, Balogh et al., Comb Chem High Throughput
Screen, 8:347 (2005)). Libraries of nitric oxide compounds have
been developed by Nitromed of Lexington, Mass.
[0223] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem.
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.).
Administration and Pharmaceutical Compositions
[0224] Molecules and compounds identified that modulate the
expression and/or function of protein are useful in treating
cancers. Modulators can be administered alone or co-administered in
combination with conventional chemotherapy, radiotherapy or
immunotherapy.
[0225] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there are a wide variety of suitable formulations of pharmaceutical
compositions of the present invention (see, e.g., Remington's
Pharmaceutical Sciences, 20.sup.th ed., 2003, supra).
[0226] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, microcrystalline cellulose, gelatin,
colloidal silicon dioxide, talc, magnesium stearate, stearic acid,
and other excipients, colorants, fillers, binders, diluents,
buffering agents, moistening agents, preservatives, flavoring
agents, dyes, disintegrating agents, and pharmaceutically
compatible carriers. Lozenge forms can comprise the active
ingredient in a flavor, e.g., sucrose, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in addition to the active ingredient, carriers known in
the art.
[0227] The compound of choice, alone or in combination with other
suitable components, can be made into aerosol formulations (i.e.,
they can be "nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0228] Suitable formulations for rectal administration include, for
example, suppositories, which consist of the packaged nucleic acid
with a suppository base. Suitable suppository bases include natural
or synthetic triglycerides or paraffin hydrocarbons. In addition,
it is also possible to use gelatin rectal capsules which consist of
a combination of the compound of choice with a base, including, for
example, liquid triglycerides, polyethylene glycols, and paraffin
hydrocarbons.
[0229] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intratumoral, intradermal, intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this invention, compositions can be administered,
for example, by intravenous infusion, orally, topically,
intraperitoneally, intravesically or intrathecally. Parenteral
administration, oral administration, and intravenous administration
are the preferred methods of administration. The formulations of
compounds can be presented in unit-dose or multi-dose sealed
containers, such as ampules and vials.
[0230] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Cells transduced by nucleic acids for ex vivo therapy
can also be administered intravenously or parenterally as described
above.
[0231] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged form. The
composition can, if desired, also contain other compatible
therapeutic agents.
[0232] Preferred pharmaceutical preparations deliver one or more
inhibitors, optionally in combination with one or more
chemotherapeutic agents, in a sustained release formulation.
Typically, the inhibitor is administered therapeutically as a
sensitizing agent that increases the susceptibility of tumor cells
to other cytotoxic cancer therapies, including chemotherapy,
radiation therapy, immunotherapy and hormonal therapy. In some
embodiments, the inhibitor can be an NO donor, including those
listed supra, a conjugate comprising NO and another agent (i.e., NO
conjugated to aspirin), or an activator of inducible nitric oxide
synthase.
[0233] In therapeutic use for the treatment of cancer, the
compounds utilized in the pharmaceutical method of the invention
are administered at the initial dosage of about 0.001 mg/kg to
about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to
about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1
mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can
be used. The dosages, however, may be varied depending upon the
requirements of the patient, the severity of the condition being
treated, and the compound being employed. For example, dosages can
be empirically determined considering the type and stage of cancer
diagnosed in a particular patient. The dose administered to a
patient, in the context of the present invention should be
sufficient to effect a beneficial therapeutic response in the
patient over time. The size of the dose also will be determined by
the existence, nature, and extent of any adverse side-effects that
accompany the administration of a particular vector, or transduced
cell type in a particular patient. Determination of the proper
dosage for a particular situation is within the skill of the
practitioner. Generally, treatment is initiated with smaller
dosages which are less than the optimum dose of the compound.
Thereafter, the dosage is increased by small increments until the
optimum effect under circumstances is reached. For convenience, the
total daily dosage may be divided and administered in portions
during the day, if desired.
[0234] The pharmaceutical preparations are typically delivered to a
mammal, including humans and non-human mammals. Non-human mammals
treated using the present methods include domesticated animals
(i.e., canine, feline, murine, rodentia, and lagomorpha) and
agricultural animals (bovine, equine, ovine, porcine).
Diagnostic Methods
[0235] The present invention also provides methods of diagnosing a
cancer, including wild-type, truncated or alternatively spliced
forms. Diagnosis can involve determining the level of expression
(transcription or translation), DNA binding activity or
intracellular localization in a patient and then comparing the
level to a baseline or range. Typically, the baseline value is
representative expression levels, DNA binding activity or
intracellular localization in a healthy person not suffering from
cancer. Variation of levels of a polypeptide or polynucleotide of
the invention from the baseline range (either up or down) indicates
that the patient has a cancer or is at risk of developing a cancer.
In some embodiments, the level of expression, DNA binding activity
or intracellular localization are measured by taking a blood, urine
or tissue sample from a patient and measuring the amount of a
polypeptide or polynucleotide of the invention in the sample using
any number of detection methods, such as those discussed
herein.
[0236] Antibodies can be used in assays to detect differential
protein expression and protein localization in patient samples,
e.g., ELISA assays, immunoprecipitation assays, and
immunohistochemical assays. In one embodiment, tumor tissue samples
are used in immunohistochemical assays and scored according to
standard methods known in the art. PCR assays can be used to detect
expression levels of nucleic acids, as well as to discriminate
between variants in genomic structure, such as insertion/deletion
mutations, truncations or splice variants. Immunohistochemistry
and/or immunofluorescence techniques can be used to detect
increased nuclear localization of proteins.
[0237] In some embodiments, overexpression of protein in a
cancerous or potentially cancerous tissue in a patient may be
diagnosed or otherwise evaluated by visualizing expression levels
and localization in situ of a polynucleotide, a polypeptide, or
fragments of either. Those skilled in the art of visualizing the
presence or expression of molecules including nucleic acids,
polypeptides and other biochemicals in the tissues of living
patients will appreciate that the gene expression information
described herein may be utilized in the context of a variety of
visualization methods. Such methods include, but are not limited
to, single-photon emission-computed tomography (SPECT) and
positron-emitting tomography (PET) methods. See, e.g., Vassaux and
Groot-wassink, "In Vivo Noninvasive Imaging for Gene Therapy," J.
Biomedicine and Biotechnology, 2:92-101(2003).
[0238] PET and SPECT imaging shows the chemical functioning of
organs and tissues, while other imaging techniques--such as X-ray,
CT and MRI--show structure. The use of PET and SPECT imaging is
useful for qualifying and monitoring the development of cancers
and/or therapy resistant cancers, including prostate cancer,
ovarian cancer, lung cancer, renal cancer, breast cancer, colon
cancer, leukemias, B-cell lymphomas, myelomas and hepatocarcinomas.
In some instances, the use of PET or SPECT imaging allows diseases
to be detected years earlier than the onset of symptoms. The use of
small molecules for labelling and visualizing the presence or
expression of polypeptides and nucleotides has had success, for
example, in visualizing proteins in the brains of Alzheimer's
patients, as described by, e.g., Herholz, K. et al., Mol Imaging
Biol., 6(4):239-69 (2004); Nordberg, A., Lancet Neurol.,
3(9):519-27 (2004); Zakzanis, K. et al., Neuropsychol Rev.,
13(1):1-18 (2003); Kung M. et al., Brain Res., 1025(1-2):98-105
(2004); and Herholz, K., Ann Nucl Med., 17(2):79-89 (2003).
[0239] A polypeptide, a polynucleotide, or fragments of either, can
be used in the context of PET and SPECT imaging applications. After
modification with appropriate tracer residues for PET or SPECT
applications, molecules which interact or bind with a transcript or
with any polypeptides encoded by those transcripts may be used to
visualize the patterns of gene expression and facilitate diagnosis
of cancers.
Compositions, Kits and Integrated Systems
[0240] The invention provides compositions, kits and integrated
systems for practicing the assays described herein using
polypeptides or polynucleotides of the invention, antibodies
specific for polypeptides or polynucleotides of the invention,
etc.
[0241] The invention provides assay compositions for use in solid
phase assays; such compositions can include, for example, one or
more polynucleotides or polypeptides of the invention immobilized
on a solid support, and a labeling reagent. In each case, the assay
compositions can also include additional reagents that are
desirable for hybridization. Modulators of expression or activity
of polynucleotides or polypeptides of the invention can also be
included in the assay compositions.
[0242] The invention also provides kits for carrying out the
therapeutic and diagnostic assays of the invention. The kits
typically include one or more probes that comprises an antibody or
nucleic acid sequence that specifically binds to polypeptides or
polynucleotides of the invention, and a label for detecting the
presence of the probe. The kits can find use, for example for
measuring the levels of protein or transcripts, or for measuring
DNA-binding activity. The kits may include several polynucleotide
sequences encoding polypeptides of the invention. Kits can include
any of the compositions noted above, and optionally further include
additional components such as instructions to practice a
high-throughput method of assaying for an effect on expression of
the genes encoding the polypeptides of the invention, or on
activity of the polypeptides of the invention, one or more
containers or compartments (e.g., to hold the probe, labels, or the
like), a control modulator of the expression or activity of
polypeptides of the invention, a robotic armature for mixing kit
components or the like.
[0243] The invention also provides integrated systems for
high-throughput screening of potential modulators for an effect on
the expression or activity of the polypeptides of the invention.
The systems typically include a robotic armature which transfers
fluid from a source to a destination, a controller which controls
the robotic armature, a label detector, a data storage unit which
records label detection, and an assay component such as a
microtiter dish comprising a well having a reaction mixture or a
substrate comprising a fixed nucleic acid or immobilization moiety.
A number of robotic fluid transfer systems are available, or can
easily be made from existing components. For example, a Zymate XP
(Zymark Corporation; Hopkinton, Mass.) automated robot using a
Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used
to transfer parallel samples to 96 well microtiter plates to set up
several parallel simultaneous STAT binding assays.
[0244] Optical images viewed (and, optionally, recorded) by a
camera or other recording device (e.g., a photodiode and data
storage device) are optionally further processed in any of the
embodiments herein, e.g., by digitizing the image and storing and
analyzing the image on a computer. A variety of commercially
available peripheral equipment and software is available for
digitizing, storing and analyzing a digitized video or digitized
optical image, e.g., using PC (Intel x86 or Pentium chip-compatible
DOS.RTM., OS2.RTM. WINDOWS.RTM., WINDOWS NT.RTM., WINDOWS95.RTM.,
WINDOWS98.RTM., or WINDOWS2000.RTM. based computers),
MACINTOSH.RTM., or UNIX.RTM. based (e.g., SUN.RTM. work station)
computers.
[0245] One conventional system carries light from the specimen
field to a cooled charge-coupled device (CCD) camera, in common use
in the art. A CCD camera includes an array of picture elements
(pixels). The light from the specimen is imaged on the CCD.
Particular pixels corresponding to regions of the specimen (e.g.,
individual hybridization sites on an array of biological polymers)
are sampled to obtain light intensity readings for each position.
Multiple pixels are processed in parallel to increase speed. The
apparatus and methods of the invention are easily used for viewing
any sample, e.g., by fluorescent or dark field microscopic
techniques.
EXAMPLES
[0246] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Regulation of Chemoresistance and Immune Resistance of B-NHL Cell
Lines by Overexpression of YY1 and Bcl-xl, Respectively: Reversal
of Resistance by Rituximab
[0247] We have recently reported that treatment of B-Non-Hodgkin's
Lymphoma (NHL) cell lines with rituximab (anti-CD20 antibody)
sensitizes the tumor cells to both chemotherapy and Fas-induced
apoptosis (Jazirehi and Bonavida, Oncogene, 24:2121-2145 (2005)).
This study investigated the underlying molecular mechanism of
rituximab-mediated reversal of immune and drug resistance.
Treatment of B-NHL cell lines inhibited the constitutively
activated NF-.kappa.B. Cells expressing dominant active I.kappa.B
or treated with NF-.kappa.B specific inhibitors were sensitized to
both drugs and FasL agonist mAb (CH-11)-induced apoptosis.
Downregulation of Bcl-.sub.XL expression via inhibition of
NF-.kappa.B activity correlated with chemosensitivity. The direct
role of Bcl-.sub.XL in chemoresistance was demonstrated by the use
of BC1-.sub.XL overexpressing Ramos cells, Ramos HA-Bcl-.sub.XL
(gift from Genhong Cheng, UCLA), which were not sensitized by
rituximab to drug-induced apoptosis. However, inhibition of
Bcl-.sub.XL in Ramos HA-Bcl-x resulted in sensitization to
drug-induced apoptosis. The role of Bcl-.sub.XL expression in the
regulation of Fas resistance was not apparent as Ramos
HA-Bcl-.sub.XL cells were as sensitive as the wild type cells to
CH-11-induced apoptosis. Several lines of evidence support the
direct role of the transcription repressor Yin-Yang 1 (YY1) in the
regulation of resistance to CH-11-induced apoptosis. Inhibition of
YY1 activity by either rituximab, the NO donor DETANONOate, or
following transfection with YY1 siRNA all resulted in upregulation
of Fas expression and sensitization to CH-11-induced apoptosis.
These findings show two complementary mechanisms underlying the
chemo-sensitization and immuno-sensitization of B NHL cells by
rituximab via inhibition of NF.kappa.B. The regulation of
chemoresistance by NF.kappa.B is mediated via Bcl-.sub.XL
expression whereas the regulation of Fas resistance by NF-B is
mediated via YY1 expression and activity. These findings show that
drug-resistant NHL tumor cells are sensitive to immune-mediated
therapeutics.
Example 2
Rituximab-Mediated Inhibition of the Transcription Repressor
Yin-Yang 1 (YY1) in NHL B Cell Lines: Upregulation of Fas
Expression and Sensitization to Fas-Induced Apoptosis
[0248] We have reported that rituximab triggers and inhibits
anti-apoptotic gene products in NHL B-cell lines resulting in
sensitization to drug-induced apoptosis (Alas et al., Clin. Cancer
Res., 8:836 (2001); Jazirehi et al., Mol. Cancer Therapy, 2:1183 (
2003); Vega et al., Oncogene, 23:3530 (2004)). This study
investigated whether rituximab also modifies intracellular
signaling pathways resulting in the sensitization of NHL cells to
Fas-induced apoptosis. Treatment of the NHL cell lines (2F7, Ramos,
and Raji) with rituximab (20 .mu.g/ml) sensitized the cells to
CH-11 (FasL agonist mAb)-induced apoptosis and synergy was
achieved. Fas expression was up-regulated by rituximab as early as
6 h post treatment as determined by flow cytometry, RT-PCR, and
Western. Rituximab inhibited both the expression and activity of
the transcription repressor Yin-Yang 1 (YY 1) that negatively
regulates Fas transcription. Inhibition of YY1 resulted in
upregulation of Fas expression and sensitization of the tumor cells
to CH-11-induced apoptosis. Downregulation of YY1 expression was
the result of rituximab-induced inhibition of both the p38MAPK
signaling pathway and constitutive NF-.kappa.B activity. The dual
roles of NF-.kappa.B and YY1 in the regulation of Fas expression
were corroborated by the use of a dominant-active inhibitor of
NF-.kappa.B (Ramos I.kappa.B-ER mutant) and YY1 siRNA,
respectively. The role of rituximab-mediated inhibition of the
p38MAPK/NF-.kappa.B/YY1 pathways, which result in both Fas
upregulation and sensitization to CH11-induced apoptosis, was
corroborated by the use of specific chemical inhibitors directed at
various components of these pathways. Rituximab-mediated
sensitization to CH-11-induced apoptosis was executed through the
Type II mitochondrial apoptotic pathway. Altogether, these findings
provide a novel mechanism of rituximab-mediated signaling by
inhibiting the p38MAPK/NF-.kappa.B/YY1 pathways and resulting in
the sensitization of B NHL to Fas-induced apoptosis. These findings
show an additional mechanism of rituximab-mediated effect in vivo
in addition to complement-dependent cytotoxicity (CDC) and
antibody-dependent cellular cytotoxicity (ADCC).
Example 3
Rituximab Diminishes the Constitutive Activity of the PI3k-Akt
Signaling Pathway in Ramos B-NHL Cells
[0249] Rituximab (chimeric anti-CD20 monoclonal antibody) is
currently being used, alone or in combination with chemotherapy, in
the treatment of B-Non Hodgkin's Lymphoma (B-NHL). We have reported
that rituximab treatment of B-NHL cell lines sensitizes the
drug-resistant tumor cells to apoptosis by various chemotherapeutic
drugs and chemosensitization was due, in large part, to the
selective inhibition of the anti-apoptotic Bcl-.sub.XL gene
product. The constitutive activation of the Akt pathway in B-NHL
results in overexpression and functional activation of Bcl-.sub.XL.
The hypothesis that the rituximab-induced inhibition of Bcl-.sub.XL
expression and chemosensitization resulted, in part, from its
inhibitory activity of the Akt pathway was tested using the
drug-resistant Ramos B-NHL cell line. Time kinetic analysis
revealed that treatment of Ramos with rituximab inhibited
phophorylation of Akt (p-Akt), but not unphosphorylated Akt and the
inhibition was first detected at 3 to 6 h post-rituximab treatment.
Similar time kinetics revealed rituximab-induced inhibition of
p-PDK1, p-Bad, p-IKK.alpha./.beta. and p-I.kappa..beta..alpha. and
no inhibition of unphosphorylated proteins. In addition, rituximab
treatment resulted in significant increase of Bcl-.sub.XL -Bad
heterodimeric complexes as compared to untreated cells. The role of
the Akt pathway in the regulation of resistance was corroborated by
the use of the Akt inhibitor, LY294002, and by transfection with
siRNA Akt. Treatment of Ramos with LY294002 resulted in inhibition
of Bcl-.sub.XL expression and sensitization of rituximab sensitive
and rituximab resistant Ramos cells to both CDDP and ADR-induced
apoptosis. Further, transfection of Ramos with Akt siRNA, but not
control siRNA, inhibited both Akt and Bcl-.sub.XL expression and
sensitized the cells to CDDP-induced apoptosis. The present
findings demonstrate for the first time that rituximab inhibits the
constitutively activated Akt pathway in Ramos and its inhibition
contributes to sensitization of the drug resistant cells to
apoptosis by chemotherapeutic drugs. The findings also identify the
Akt pathway as target for therapeutic intervention in the reversal
of rituximab and drug resistant B-NHL.
Materials and Methods
Reagents
[0250] RPMI-1640, opti-MEM and fetal bovine serum (FBS) were
purchased from Gibco (Grand Island, N.Y., USA). Rituximab (stock,
10 mg/mL) was obtained commercially. Rituximab (Fab)'.sub.2 was a
generous gift from Dr. Chin (Idec Biogen, San Diego). LY294002,
anti Lyn, anti-phospho Lyn (Tyr507), anti-IKK.alpha./.beta.,
anti-IKb.alpha./.beta., anti-IKK.beta., anti-Bcl-.sub.XL, anti-Bad,
anti-phospho-Bad (Ser136), anti-phopho PDK1, anti-human phospho-Akt
(Ser473, Thr308), anti-Akt, and anti-.beta.-actin antibodies were
obtained from Cell Signaling Technology (Beverly, Mass., USA). Akt
siRNA and control scramble siRNA were purchased from Sigma (Mo.,
USA). Protein A-agarose was purchased from Pierce (Rockford,
Ill.).
Cell Culture
[0251] The CD20+human Burkitt's lymphoma B-cell line Ramos was
obtained from ATCC (Manassas, Va., USA). Cells were cultured in 50
ml culture flasks (Costar, Cambridge, Mass.) at 37.degree. C. in an
atmosphere of 5% CO.sub.2 in RPMI 1640 supplemented with 10% (v/v)
heat-inactivated FBS (to ensure the absence of complement),
penicillin/streptomycin, non essential amino acid and
2-mercaptoethanol. The Ramos RR1 clone was generated in our
laboratory following culture of Ramos in the presence of increasing
concentrations of rituximab for 10 weeks and clones were isolated
by limiting dilution (Jazirehi et al., 2006).
Drug Pretreatment
[0252] Ramos B-NHL cells (1.times.10.sup.6 cells/ml) were grown in
complete medium in 50 ml tissue culture flasks and were treated
with a previously established optimal concentration (20 ug/ml) of
rituximab for 18 h. The cells were then washed and fresh medium was
added and seeded into 12 well plates (Costar, Cambridge, Mass.).
Indicated concentrations of drugs (CDDP, ADR) were then added, and
the cells were incubated for another 24 h for maximal cytotoxicity.
At the end of the incubation period, the cells were harvested and
subjected to propidium iodide (PI) (Molecular Probes, Leiden,
Netherlands) staining according to the specifications of the PI
staining kit (Roche Diagnostics Corporation, Indianapolis, Ind.)
and evaluated by flow cytometric analysis. LY294002 was dissolved
in DMSO (Sigma-Aldrich) and added up to 50 uM.
Analysis of Apoptosis
[0253] DNA fragmentation was detected by flow cytometric analysis
after propidium iodide staining (Alas, S. et al., Cancer Res.,
61:5137-44 (2001)). Cells with fragmented DNA were considered as
apoptotic. Fixation and staining of the cells were performed
according to standard protocols. Briefly, samples containing
1.times.10.sup.6 cells were collected, resuspended in ice-cold PBS
and fixed with ethanol (70% v/v). After 50 min incubation at
4.degree. C., cells were centrifuged and incubated with 50 ug/ml of
PI for 20 min at 37.degree. C. Fluorescence was measured on a
FACScan flow cytometer (BD Immnunocytometry Systems) within 1 h. A
minimum of 10,000 events was collected on each sample and acquired
in listmode by a PC Pentium computer. Cellular debris were excluded
from analysis by raising the forward scatter threshold, and the DNA
content of the intact nuclei was recorded on a logarithmic scale.
The percentage of apoptotic cells is represented as the percentage
of hypodiploid cells accumulated at the sub-GO phase of the cell
cycle.
Western Blotting
[0254] After stimulation, cells (2.times.10.sup.6 cells) were
collected and resuspended with lysis buffer (20 mM Tris-HCl (pH
8.0), 2 mM EDTA, 3% NP-40, 100 mM NaCl, 50 mM NaF, 1 uM PMSF, 1 uM
V04, 5 ug/ml aprotinin, 5 ug/ml leupeptin). After 30 min of
incubation on ice, samples were centrifuged at 10,000.times.g, for
10 min at 4.degree. C. to remove cellular debris. The concentration
of the protein in the resulting supernatant was measured using the
Bio-Rad protein assay reagent and equalized with SDS-PAGE buffer
(0.125 M Tris-HCI pH 6.8, 4% SDS, 20% glycerol, 10%
2-mercaptoethanol, bromophenolblue).Then equal amounts of proteins
were separated using sodium dodecylsulphate-polyacrylamide gel
electrophoresis (10-12% SDS-PAGE) with a Mini-Protean system
(Bio-Rad, Hercules, Calif., USA) and electrophoretically
transferred to nitrocellulose membranes. The membranes were blocked
for 30 min with 5% non-fat dry milk diluted in 0.05% Tween-20
Tris-buffered saline at room temperature and incubated overnight
with the primary antibodies. After incubation with the primary
antibody against Akt (1:300), Bad, PDK1, (1:500), phospho-specific
antibodies against phospho-Akt (Ser 473, Thr 308) (1:300),
phospho-PDK1 and phospho-Bad (Ser136) (both 1:300) for overnight,
the membranes were washed and incubated with HRP-coupled secondary
antibodies (Cell Signaling, Mass., USA). Immunoreactive bands were
detected using an enhanced chemiluminiscence system (Amersham
Pharmacia Biotech, UK).
Immunoprecipitation
[0255] Ramos cells (2.times.10.sup.6 cells) were collected and
resuspended with lysis buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA,
3% NP-40, 100 mM NaCl, 50 mM NaF, 1 uM PMSF, 1 uM V04, 5 ug/ml
aprotinin, 5 ug/ml leupeptin). After 30 min of incubation on ice,
samples were centrifuged at 10000.times.g, for 10 min at 4.degree.
C. to remove cellular debris. The concentration of the protein in
the resulting supernatant was measured using the Bio-Rad protein
assay reagent and equalized with RIPA buffer. One mg of total
lysate was incubated with 5 .mu.l protein A agarose beads (Pierce,
Rockford, Ill.), centrifuged and the supernatant was recovered. The
supernatant was mixed with 15 .mu.l of protein A agarose and 2
.mu.g Bcl-.sub.XL mouse monoclonal antibody or normal mouse IgG-1
at 4.degree. C. for 4 h. Following centrifugation, the beads were
washed 4.times. with RIPA buffer and the beads were boiled in 50
.mu.l Laemmli buffer and subjected to SDS-polyacrylamide gel
electrophoresis and blotted with rabbit anti-Bad antibody. After
overnight incubation with the primary antibody, the membranes were
washed and incubated with HRP-coupled secondary goat anti-rabbit
antibody (Cell Signaling, Mass., USA). Immunoreactive bands were
detected using an enhanced chemiluminiscence system (Amersham
Pharmacia Biotech, UK).
Transfection of Ramos with Akt siRNA
[0256] Ramos B-NHL cells were cultured in 1 ml of Opti-MEM medium
without antibiotics and FBS. Transfections were performed using
Xtreme GENE reagent (Roche Diagnostics Corporation, Indianapolis,
Ind.), Akt siRNA (Sigma, Mo., USA) and control scramble siRNA
(Sigma, Mo., USA) according to the manufacturers' instructions.
Briefly, 4 ul of Akt siRNA or a scramble control siRNA solution
mixed with 96 ul of opti-MEM was incubated with 10 ul of the
transfection reagent in 90 ul of serum-free Opti-MEM medium for 15
min to facilitate complex formation. The resulting mixture was
gently added to Ramos cells cultured in a 12 well plate with 1 ml
of medium. Akt, phospho-Akt and Bcl-.sub.XL levels were determined
by Western blotting. To determine Akt siRNA-induced sensitization
to CDDP-induced apoptosis, following transfection the cells were
treated with CDDP for 24 h, then fixed and permeabilized for PI
staining, and analyzed by flow cytometry as described above.
Statistical Analysis
[0257] Assays were set up in triplicates and the results were
expressed as the mean.+-.SD. Statistical analysis and P value
determinations were done by two-tailed paired t test with a
confidence interval of 95% for determination of the significance of
differences between the treatment groups. P<0.05 was considered
to be significant.
Example 3A
The Effects of Rituximab Treatment on the Activity of the Akt
Pathway
[0258] Rituximab treatment of B-NHL cell lines results in
inhibition of the survival Raf-1/MEK/ERK1/2 and the NF-.kappa.B
signaling pathways both of which led to the selective inhibition of
the expression of the anti-apoptotic gene product Bcl-.sub.XL. This
results in the sensitization of drug-resistant B-NHL tumor cells to
apoptosis by various chemotherapeutic drugs. Here, other signaling
pathways that also regulate Bcl-.sub.XL expression and/or activity
were explored as to whether they may also be inhibited by
rituximab. The Akt pathway which is constitutively activated in
many cancers in part by Src kinases and PI3K (Inabe, K. et al.,
Blood, 99:584-9 (2002); Tedder, T. et al., Biochemical Society
Transactions, 30:807-811 (2002)) has been shown to regulate
Bcl-.sub.XL transcriptional and post-transcriptional expression and
activity (Hayakawa, J. et al., Cancer Res., 60:5988-94 (2000);
Weintraub, S. et al., Cancer Cell, 5:3-4 (2004); Castilla, C. et
al., Endocrinology [Epub ahead of print] (2006)).
[0259] Ramos cells were treated with rituximab (20 .mu.g/ml) for
different time periods (0-24 h) and cell lysates were prepared for
analysis of various proteins of the Akt signaling pathway
(non-phosphorylated and phosphorylated proteins) by western. Time
kinetic analyses revealed that rituximab treatment of Ramos
inhibited p-Lyn, p-Akt (Ser473 and Thre308), p-PDK1 and p-Bad while
there was no detectable inhibition of non-phosphorylated proteins.
The inhibition was first observed at 3-6 h post rituximab treatment
(FIG. 1A). The rituximab-mediated inhibition of p-Akt was a direct
effect of rituximab interaction with CD20 as treatment of Ramos
with Fc-devoid Rituximab (Fab').sub.2 also inhibited p-Akt like
rituximab. This finding ruled out any contribution of the Fc in
rituximab-mediated cell signaling (FIG. 1B).
[0260] The PI3K-Akt signaling pathway regulates the transcriptional
expression of Bcl-.sub.XL via IKK, I.kappa.B and NF-.kappa.B
activation (Sugimori, K. et al., J Bone Miner Metab., 23:411-9
(2005)). Whether rituximab treatment of Ramos could inhibit the
phosphorylation of these gene products was next investigated. This
was analyzed by examining total cell lysates of Ramos cells that
were treated with rituximab for different time periods (1-24 h) and
thereafter they were subjected to analysis by western using
phospho-specific and non-phospho-specific antibodies. Treatment of
Ramos cells with rituximab diminished the levels of
p-IKK.alpha./.beta. and p-I.kappa..beta..alpha., but not the levels
of non-phosphorylated proteins and the inhibition was first
detected at 3-6 h post-rituximab treatment and significant
inhibition was observed after 24 h (FIG. 1C).
Example 3B
Decreased Bcl-.sub.xL Activity and Expression by Rituximab-mediated
Inhibition of the Akt Pathway
[0261] The above findings demonstrated that rituximab treatment of
Ramos cells diminishes the activity of the constitutively activated
Akt pathway. The Akt signaling pathway regulates Bcl-.sub.XL
activity via phosphorylation of Bad, which normally dissociates
from Bcl-.sub.XL and dissociated Bcl-.sub.XL can thus exert its
anti-apoptotic activity. Likewise, phosphorylation of Bad can also
promote cell survival (Datta, S. et al., Genes & Development,
13:2905-2927 (1999); Hayakawa, J. et al., Cancer Res., 60:5988-94
(2000)). We next investigated whether the constitutive activation
of the Akt pathway in Ramos cells could lead to phosphorylation of
Bad and therefore, unbound cytosolic Bcl-.sub.XL which can exert
its anti-apoptotic activity.
[0262] Treatment of Ramos with rituximab, which inhibited
phospho-Bad (FIG. 1A), was found to significantly enhance the
physical association of Bad and Bcl-.sub.XL. This was determined by
treating Ramos cell lysates with anti-Bcl-.sub.XL antibody and the
immuno-precipitated complexes were analyzed for Bad by western. It
is clearly shown in FIG. 2A that treatment of Ramos cells with
rituximab significantly augmented the association of Bad with
Bcl-.sub.XL as compared to untreated cells. These findings
demonstrate that treatment of Ramos with rituximab inhibited the
Akt signaling pathway and enhanced the complex formation of
non-phosphorylated Bad with Bcl-.sub.XL and hence, reducing the
anti-apoptotic activity of Bcl-.sub.XL.
[0263] Treatment of Ramos cells with rituximab significantly
inhibited Bcl-.sub.XL expression via inhibition of the ERK 1/2 and
NF-.kappa.B pathways confirming our previous findings (Jazirehi, A.
et al., Cancer Res., 65:264-76 (2005)) (FIG. 2B). We examined the
direct role of the Akt pathway in the regulation of Bcl-.sub.XL
expression. Ramos cells were treated with the Akt specific
inhibitor, LY294002, and cell lysates were examined for levels of
Bcl-.sub.XL . Treatment of Ramos cells with LY294002 significantly
inhibited Bcl-.sub.XL expression and mimicked rituximab mediated
inhibition of Bcl-.sub.XL expression (FIG. 2B). These findings
suggested that the Akt pathway may be involved in
rituximab-mediated inhibition of Bcl-.sub.XL function.
Example 3C
Participation of Rituximab-mediated Inhibition of the Akt Pathway
in the Sensitization of Ramos Cells to Drug-induced Apoptosis
[0264] Two approaches were examined to determine the role of the
AKT pathway in chemosensitization, namely, (i) the use of a
pharmacologic specific Akt inhibitor, Ly294002, and (ii) inhibition
of Akt by siRNA.
(i) Inhibition of the Akt pathway by LY294002 mimics
rituximab-mediated sensitization of Ramos cells to both CDDP and
ADR-induced apoptosis.
[0265] Rituximab-mediated inhibition of the ERK1/2 and NF-.kappa.B
pathways and Bcl-.sub.XL expression can overcame drug-resistance
and sensitize the tumor cells to apoptosis by various
chemotherapeutic drugs. Here, we show, rituximab and
rituximab-Fab'.sub.2 treatment of Ramos cells for 24 h sensitize
the cells significantly to CDDP-induced apoptosis, respectively,
and the sensitization was a function of the antibody concentration
used. Treatment with single agent had no cytotoxic activity (FIG.
3A,B). By comparison, treatment of Ramos cells with various
concentrations of LY294002 also sensitized the cells to
CDDP-induced apoptosis and the sensitization was a function of the
LY294002 concentration used (FIG. 3C). Similar findings were
observed for LY294002-mediated sensitization of Ramos cells to
ADR-induced apoptosis (FIG. 3D). In addition to the wild-type, we
have also examined the effect of LY294002 in the sensitization of
rituximab-resistant Ramos clone (Ramos RR1). Treatment of Ramos
RR1with rituximab did not sensitize the cells to CDDP-induced
apoptosis (FIG. 3E); however, treatment with LY294002 resulted in
significant sensitization of Ramos RR1 to CDDP-induced apoptosis
(FIG. 3F).
(ii). Sensitization of Ramos cells to drug-induced apoptosis by
transfection with Akt siRNA.
[0266] The direct role of the Akt pathway in the regulation of drug
resistance was tested by transfecting Ramos cells with Akt siRNA as
described in methods. The specificity of transfection was
determined by using a control siRNA. Treatment of Ramos cells with
Akt siRNA resulted in significant inhibition of p-Akt and Akt in a
time-dependent manner. The inhibition of Akt by Akt siRNA resulted
in inhibition of Bcl-.sub.XL expression; however, transfection with
control siRNA had no effect (FIG. 4A). The cells tranfected with
siRNA were then examined for sensitivity to CDDP-induced apoptosis.
Since the findings above demonstrated that Ly294002-induced
inhibition of Akt sensitized the cells to both CDDP and ADR-induced
apoptosis, we tested whether inhibition of Akt by siRNA could also
sensitize Ramos cells to drug-induced apoptosis. Indeed, treatment
with Akt siRNA, but not with siRNA control, sensitized the cells to
CDDP-induced apoptosis (FIG. 4B).
[0267] Altogether, the above findings demonstrate that
rituximab-induced inhibition of the Akt pathway contributes to
rituximab-mediated sensitization of rituximab sensitive and
rituximab resistant Ramos cells to drug-induced apoptosis.
[0268] The present findings demonstrate for the first time that
treatment of the B-NHL cell line Ramos with rituximab resulted in
significant inhibition of the constitutively activated PI3k/Akt
signaling pathway. Rituximab-mediated inhibition of this pathway
resulted in both inhibition of Bcl-.sub.XL activity through the
increased formation of complexes between Bad and Bcl-.sub.XL as
well as down regulation of Bcl-.sub.XL expression. Rituximab
sensitized the drug-resistant Ramos cells to apoptosis by
chemotherapeutic drugs such as CDDP and adriamycin. The role of the
Akt pathway in the regulation of chemoresistance was corroborated
by both the use of the PI3K inhibitor Ly294002 and by the use of
silencer RNA for Akt, both of which inhibited Bcl-.sub.XL
expression and sensitized the tumor cells to drug-induced
apoptosis. These findings establish in B-NHL the involvement of the
Akt pathway in chemoresistance and whose inhibition by rituximab
results in chemosensitivity. In addition, the present findings
identify the Akt pathway as a novel target for therapeutic
intervention and inhibitors of this pathway can be used alone or in
combination with drugs in the treatment of rituximab and drug
resistant B-NHL.
[0269] Aberrant activation of the PI3K-Akt pathway has been widely
implicated in many cancers. PI3K is a signaling component
downstream of growth factor receptors tyrosine kinases (RTKs)
(Cantley, L., Science, 296:1655-7 (2002)). The PI3K-Akt signaling
pathway regulates many normal cellular processes including cell
proliferation, survival, growth and motility processes that are
critical for tumorigenesis (Vivanco, I. et al., Nat Rev Cancer,
2:489-501 (2002)). Hyperactivation of the PI3K-Akt pathway is often
genetically selected during tumorigenesis and the normal cellular
functions regulated by this pathway are recruited to promote
proliferation and survival of cancer cells. The PI3K-Akt pathway is
a key regulator of cell survival through multiple downstream
targets. PI3K phosphorylates PIP2 at the 3' position on the
inositol ring and converts PiP2 to PiP3. Subsequently, PIP3
recruits other downstream molecules, particularly the
serine-threonine kinases Akt and PDK-1 via binding to their
pleckstrin homology (PH) domains. Akt is partially activated
through phosphorylation at serine 308 in its activation loop by
PDK-1. Additional phosphorylation of serine 473 in the C-terminus
of Akt results in its full activation. Ramos cells exhibit
constitutively activated signaling of the Akt pathway. Rituximab
treatment inhibited both pAkt (ser473) and pAkt (Thr308) 3-6 hours
post treatment and the inhibition was augmented as a function of
time up to 24 hours. There was no inhibition of non phosphorylated
Akt. The inhibition pPDK1 by rituximab followed the same time
kinetics as those for Akt. Akt can phosphorylate the Bcl-2 family
member Bad, causing its sequestration from the mitochondrial
membrane by 14-3-3 protein (Datta, S. et al., Genes &
Development, 13:2905-2927 (1999)). Bad is a member of the family of
Bcl-2 proteins functioning as apoptosis regulatory factors. Bad in
its unphosphorylated form binds to and inactivates anti-apoptotic
proteins such as Bcl-2 and Bcl-.sub.XL leading to its pro-apoptotic
function. Recently, Akt has been shown to phosphrylate Bad at
serine 136 which allows Bad to dissociate from the
Bcl-2/Bcl-.sub.XL complex and loses its pro-apoptotic function
(Henshall, D. et al., J Neurosci., 22:8458-65 (2002); Kamada, H. et
al., J Cereb Blood Flow Metab. [Epub ahead of print] (2006)).
Inhibition of pBad was initially observed at 6 hour post rituximab
treatment in the absence of inihibition of non-phosphorylated Bad.
The inhibition of pBad in Ramos resulted in the augmented
association of Bad with Bcl-.sub.XL to form more complexes.
Complexes of Bad and Bcl-2 could not be detected in Ramos since
these cells are deficient in Bcl-2 expression and we have
previously confirmed this finding (Jazirehi, A. et al., Cancer
Res., 64:7117-26 (2004)).
[0270] Our data showed that in Ramos, aside from the proteins
responsible for the activation of the Akt pathway, IKK and IKB were
constitutively activated. We have previously reported that
rituximab treatment sensitized NHL cells to drug induced-apoptosis
via inhibition of NF.kappa.B. A cross talk between the PI3K and the
NF.kappa.B pathway has been previously reported in a number of
systems (Yin, D. et al., J Neuroimmunol., 174:101-7 (2006);
Fishman, P. et al., Arthritis Res Ther, 8:R33 (2006)). As shown in
this study, treatment with rituximab inhibited the phosphorylation
of IKK and I.kappa.B.alpha. indicating that rituximab inhibits the
anti-apoptotic effect regulated by NF.kappa.B. Considerable
controversy remains regarding the involvement of Akt in
signal-induced IKK activation. Studies by Ozes, O. et al., Nature,
401:82-5 (1999) indicated that Akt was required for TNF.alpha. or G
protein activation induced NF.kappa.B activation by directly
phosphorylating and activating IKK.alpha. in tumor cell lines.
Chandramohan, V. et al., J Immunol., 172:5522-7 (2004) showed in
B-lymphoma cells generated from mice that inhibition of the
PI3K/Akt signaling pathway resulted in decreased level of
NF.kappa.B. Thus, it is possible that Akt phosphorylates IKK in a
cell context dependent and stimulation-dependent manner.
[0271] The direct role of the activated Akt pathway in the
regulation of resistance was independently demonstrated by the use
of a pharmacologic inhibitor and by siRNA. Both inhibited
Bcl-.sub.XL expression and sensistized the tumor cells to
drug-induced apoptosis. Ly294002 has long been used as a selective
inhibitor of PI3K-mediated phosphorylation of Akt (Poh, T. et al.,
Cancer Res., 65:6264-74 (2005)). The biological activity mediated
by Ly294002 and its relevance to apoptosis has been attributed to
its effect on the PI3K/Akt survival network. Ly294002 is a
flavonoid therapeutic; alone it has anti-proliferative and
pro-apoptotic activities (Wetzker, R. et al., Curr Pharm Des.,
10:1915-22 (2004)). Administration of Ly294002 in mice bearing
human xenograft inhibited tumor growth and induced apoptosis
(Semba, S. et al., Cancer Res., 8:1957-63 (2002); Fan, Q. et al.,
Cancer Res., 63:8930-8 (2003)). The combinational treatment with
cytotoxic drugs enhances the effectiveness of the treatment
(Wetzker, R. et al., Curr Pharm Des., 10:1915-22 (2004)). Liu, X.
et al., Mol Cancer Ther., 5:494-501(2006) examined the role of the
PI3K/Akt pathway in resistance to drug-induced apopotosis. They
showed that the inhibitior LY2940002 which inhibits PI3K sensitized
breast cancer cell lines to cerulenin induced apopotosis. Nuutinen,
U. et al., Exp Cell Res., 312:322-30 (2006) reported recently that
inhibition of PI3K or Akt markedly enhanced dexamethasome induced
apoptosis in a human follicular lymphoma cell line. These findings
are consistent with our present findings demonstrating that
treatment of Ramos with LY294002 sensitizes cells to both CDDP and
ADR-induced apoptosis. In addition, we demonstrate that the
treatment of rituximab-resistant RR1 cells, which cannot be
chemosensitized by rituximab, can be sensitized by LY294002 to
drug-induced apoptosis. This finding is of potential therapeutic
application in the treatment of drug and rituximab-resistant tumor
cells. In addition to LY294002, knockdown of Akt by siRNA
significantly reduced Bcl-.sub.XL expression as well as both AKT
and p-Akt and the cells were sensitized to CDDP-induced apoptosis.
Clinically, knockdown of Akt by anti-sense or siRNA significantly
reduced tumor cell growth and invasiveness and induced cell growth
arrest and apoptosis in tumor cells overexpressing Akt (Cheng, J.
et al., Proc Natl Acad. Sci USA, 93:3636-41 (1996); Remy, I. et
al., Mol Cell Biol., 24:1493-504 (2004); Tabellini, G. et al., J
Cell Physiol., 202:623-34 (2005)).
[0272] Bcl-.sub.XL, a member of the Bcl-2 family, exerts an
anti-apoptotic effect on lymphocytes. Zhao, W. et al., Blood,
103:695-7 (2004) investigated the clinical significance of
Bcl-.sub.XL expression in patients with follicular lymphoma using
real time quantitative RT-PCR. Lymph node sections and laser
micro-dissected lymphoma cells from 27 patients were analyzed. The
gene was overexpressed in patients with follicular lymphoma at a
higher level in micro-disected lymphoma cells. A high Bcl-.sub.XL
level was significantly associated with progression and with the
international prognostic index indicating high risk. Moreover,
Bcl-.sub.XL gene overexpression was linked to short overall
survival (Zhao, W. et al., Blood, 103:695-7 (2004)). Bcl-.sub.XL is
abundantly expressed in lymphoma (Xerri, L. et al., Br J Haematol.,
92:900-6 (1996)) and protects the cells from apoptosis induced by
DNA damaging agents. An inverse correlation was found between
levels of Bcl-.sub.XL and sensitivity to 122 standard cancer agents
has been established (Amundson, S. et al., Cancer Res., 60:6101-10
(2002)). In the present study, there was a strong correlation
between Bcl-.sub.XL inhibition by rituximab treatment and
chemosensitization.
[0273] Various mechanisms contribute to activation of Akt pathways
in tumors, including perturbation of upstream PTEN and PIP3
(Vivanco, I. et al., Nat Rev Cancer, 2:489-501 (2002)). Others
include autocrine or paracrine stimulation of receptor tyrosine
kinases and overexpression of growth factor receptors and/or Ras
activation. It has been shown that Akt is activated constitutively
by active Ras and Src (Datta, S. et al., Genes & Development,
13:2905-2927 (1999); Lin, R. et al., J Biol Chem., 272:31196-202
(1997)). Thus, in Ramos cells, we have reported that rituximab
inhibits the Src kinase pLyn and it is possible that this
inhibition is involved in the downstream inhibition of the Akt
pathway by rituximab. The PTEN tumor suppressor is a negative
regulator of the Akt pathway. The loss of PTEN function leads to an
elevated concentration of the PIP3 substrate and results in
constitutive activation of downstream components of the PI3K
pathway, including the Akt and mTOR kinases (Di Cristofano, A. et
al., Cell, 100:387-90(2000)). PTEN is a prominent member and a
mechanism leading to selective Akt inhibition. PTEN, as a result of
its decreased activity, leads to selective Akt activation.
Targeting of Akt, directly or indirectly, inhibits cell
proliferation, promote apoptosis, and/or increase sensitivity to
chemotherapy (Tachiiri, S. et al., Jpn J Cancer Res., 91:1314-8
(2000)). The loss of PTEN expression has been reported in a variety
of cancer including lung, breast, prostate, colon, indometrium, and
glioblastoma and has been shown to incur through mutation,
deletion, and epigenetic mechanisms (Rennie, P. et al., Cancer
Metastasis Rev., 17:401-9 (1998); Marsit, C. et al., Hum Pathol.,
36:768-76 (2005); Haiman, C. et al., Cancer Epidemiol Biomarkers
Prev., 15:1021-5 (2006)). There are relatively a few studies
exploring PTEN abnormalities in lymphoma. Preliminary findings
indicate that rituximab treatment of Ramos resulted in PTEN
induction.
Example 3D
Exemplary Targets for Therapeutic Intervention
[0274] The present findings demonstrate that rituximab inhibits the
constitutively activated Akt pathway in the Ramos B-NHL cells and
results in inhibition of both Bcl-.sub.XL activity and expression
leading to reversal of drug-resistance. A schematic diagram
summarizing the findings of this study is illustrated in FIG. 5.
Our findings indicated that modulation of the PI3K/Akt pathway and
components of the Akt signal transduction pathway by rituximab are
responsible, in part, for chemosensitization. Various gene products
of this pathway are potential targets for therapeutic intervention
particularly that Akt signaling promotes cell survival,
proliferation and invasion. Blocking this pathway could impede the
proliferation of tumor cells by increasing sensitivity of tumor
cells to undergo apoptosis in response to other cytotoxic agents.
Several inhibitors have been the subject of several investigations
to intervene in the Akt pathway for tumor cell sensitization
(Cheng, J. et al., Oncogene, 24:7482-92 (2005)). Likewise, this
study identifies the Akt pathway and each of its component members
as set forth on FIG. 5 as targets for therapeutic intervention in
the treatment of drug-resistant and/or rituximab-resistant NHL.
Hyper-activation of the Akt pathway is thus implicated in the
pathogenesis of NHL and the development of
drug/rituximab-resistance. Accordingly, as hyper-activation of this
pathway may serve as a quite useful prognostic indicator in
patients who are refractory to conventional therapies, methods of
monitoring such via the activity, activation, expression, or
cellular levels of any one or more of the components of the
pathways of FIG. 5 can be useful as prognostic indicator.
Example 4
Sensitization of Rituximab-sensitive and Rituximab-resistant B-NHL
Cell Lines/Clones to TRAIL-induced Apoptosis by Bortezomib and
NF.kappa.B Inhibitors
[0275] Patients with B-NHL respond initially to conventional
chemotherapy and/or to immunotherapy with rituximab (alone or in
combination with chemotherapy). However, patients develop
resistance to these modalities and novel approaches are needed.
TRAIL is a cytotoxic molecule that exerts selective anti-tumor
cytotoxic activity with minimal toxicity to normal tissues.
Further, TRAIL or agonist monoclonal antibody (mAb) to TRAIL
receptors, DR4 and DR5, are currently being tested clinically. The
present study investigated the sensitivity of B-NHL cell lines to
TRAIL-mediated apoptosis using the AIDS-related NHL (ARL) B-cell
line, 2F7, and the B-NHL cell lines, Ramos and Daudi. Also, to
recapitulate various aspects of acquired rituximab-resistance, we
have generated rituximab-resistant (RR) clones from the parental
wild type (wt) cells. Rituximab failed to chemo-sensitize the RR
clones and the clones exhibited higher resistance to various drugs
(e.g., CDDP, VP-16, ADR, Vincristine, Taxol) and to TRAIL (1-250
ng/ml-18 h) compared to the wt cells as analyzed by DNA fragment on
assay. The findings demonstrate that the wild type and RR1 cells
were resistant to TRAIL-mediated apoptosis at a wide range of TRAIL
concentrations. We then examined means to reverse B.kappa.TRAIL
resistance. We and others have reported that inhibition of
NF.kappa.B activity can sensitize TRAIL-resistant tumor cells to
TRAIL-induced apoptosis. Hence, we examined the effect of the
proteasome and NF.kappa.B inhibitor, Bortezomib (Velcade), Bay
11-7085 and the specific NF.kappa.B inhibitor DHMEQ (Kikuchi et.
al, Cancer Research, 63:107 (2003)). Pretreatment of the NHL tumor
cells with Bortezomib, Bay 11-7085 or DHMEQ for 2 h followed by
treatment with TRAIL for 18 h resulted in significant augmentation
of apoptosis and synergy was achieved. Both the rituximab-sensitive
and rituximab-resistant tumor cells were sensitized by these
inhibitors, though higher concentrations were required for
sensitization of the RR clones. Interestingly, detailed analysis of
the signaling pathways in the RR clones revealed constitutive
hyper-activation of the NF.kappa.B survival pathway leading to
over-expression of anti-apoptotic gene products Bcl-2, Bcl-.sub.XL
and Mcl-1. Based on the findings, patients with resistant B-NHL can
be treated with combination of TRAIL/anti-DR4 or DR5 mAb and
NF.kappa.B inhibitors. Alternatively, these patients can be treated
with agents that up-regulate TRAIL expression on host effectors
(e.g., T cells, NK cells) in combination with NF.kappa.B
inhibitors.
Example 5
Reversal of Rituximab-resistant AIDS-B-NHL Clone to
Chemotherapeutic Drug-induced Apoptosis by Bortezomib and DHMEQ
[0276] The mechanisms underlying the failure of B-NHL cancer
patients to respond to treatment with rituximab, alone or in
combination with chemotherapy, are not known. In efforts to address
this issue, we have generated rituximab-resistant clones of the
AIDS NHL cell line, (2F7RR). Recent findings have demonstrated that
treatment of the wild type (wt) 2F7 with rituximab sensitized the
tumor cells to various chemotherapeutic drug-induced apoptosis.
Chemosensitization was the result of rituximab-mediated inhibition
of the p38 MAPK signaling pathway and the selective inhibition of
the anti-apoptotic Bcl-2 gene product (Vega et. al., Oncogene,
23:4993 (2004)). Analysis of one clone, 2F7RR1, revealed that the
cells have diminished surface CD20 expression and failed to respond
to CDC and to apoptosis following cross-linking. In addition, the
cells were resistant to rituximab-mediated chemosensitization. In
contrast to wt2F7, molecular analysis of the 2F7RR1 clone revealed
that rituximab failed to inhibit p-Lyn, p38-MAPK, Bcl.sub.XL, and
Bcl-2. In addition, rituximab failed to inhibit the transcription
factors NF-.kappa.B, YY1, SP-1, and STAT3. Noteworthy, 2F7RR1
exhibited higher resistance to drug-induced apoptosis compared to
wt2F7 and showed overexpression of Bcl-2. Previous findings with
the wt2F7 demonstrated that Bcl-2 was responsible for
chemoresistance. Accordingly, we examined whether inhibition of
Bcl-2 in 2F7RR1 can reverse chemoresistance. Since Bcl-2 is under
the transcriptional regulation of NF.kappa.B, we examined the
effect of the NF-.kappa.B inhibitors Bortezomib and DHMEQ (Kikuchi,
et al., Cancer Research, 63:107 (2003)). The findings revealed that
treatment of 2F7RR1 with these inhibitors resulted in the reversal
of resistance to a number of chemotherapeutic drugs (examples:
taxol, vincristine, ADR, CDDP, VP16, etc.). The extent of
chemo-sensitization by Bortezomib and DHMEQ was comparable. These
studies present evidence that rituximab and drug-resistant tumor
cells may be sensitized to chemotherapeutic drug-induced apoptosis
via inhibition of NF.kappa.B or Bcl-2. These findings also indicate
that Bortezomib and DHMEQ can be used to treat rituximab and
drug-resistant AIDS-B-NHL and other cancers.
Example 6
New Targets Identified for Therapeutic Intervention in the Reversal
of Rituximab and Drug Resistant AIDS-B-NHL
[0277] Rituximab has been used in the treatment of B Non-Hodgkin's
Lymphoma (NHL) with significant clinical responses. However, a
subset of patients fails to respond to treatment with rituximab
used alone or in combination with chemotherapy. The mechanism by
which B-NHL patients resist treatment is not known. We have
reported that treatment of the AIDS B-NHL cell line 2F7 with
rituximab resulted in significant inhibition of the p38 MAPK
pathway and inhibition of Bcl-2 expression concomitantly with
chemoresistance. The pivotal role of Bcl-2 in chemoresistance was
demonstrated by various methods as inhibition of Bcl-2 expression
or activity which sensitized the tumor cells to drug-induced
apoptosis (Vega et al., Oncogene, 23(20):3530-40 (2005)). In order
to examine the mechanism of NHL resistance to rituximab, we have
developed in the laboratory rituximab-resistant clones of 2F7
(2F7RR) and have compared their response with the wild type to
rituximab treatment alone and in combination with chemotherapeutic
drugs. Unlike the wild type 2F7, rituximab treatment failed to
sensitize 2F7 RR1 to drug-induced apoptosis, failed to modulate the
p38MAPK/NF-.kappa.B/YY1/STAT3 signaling pathways, did not inhibit
Bcl-2 expression. We examined the effect of various chemical
inhibitors of this signaling pathway on chemosensitization. We
demonstrate that treatment with the proteasome inhibitor Bortezomib
or the NF-.kappa.B inhibitor DHMEQ significantly sensitized 2F7-RR1
cells to drug (CDDP, vincristine, adriamycin, VP 16, taxol)-induced
apoptosis. These inhibitors also resulted in the inhibition of
NF-.kappa.B, YY1 and downregulated Bcl-2 expression. These findings
demonstrate that rituximab and drug-resistant clone 2F7-RR1 can be
sensitized to reverse chemoresistance. The findings also identify
intracellular targets whose modification can reverse resistance.
Such targets include the p38 MAPK pathway, the transcription
factors NF-.kappa.B, YY1, or STAT3 and also inhibitors of Bcl-2
expression and/or activity. These findings also suggest that
combination treatment of currently used drugs such as Bortezomib
and chemotherapeutic drugs have a potential for the treatment of
rituximab and drug-resistant AIDS-B-NHL.
Example 7
Rituximab-mediated Inhibition of the Akt Pathway and Upregulation
of PTEN Expression in Ramos B-NHL Leading to Chemosensitization to
Drug-induced Apoptosis
[0278] Rituximab treatment of drug resistant B-NHL cell lines
sensitizes the tumor cells to drug (e.g. CDDP, VP16, Taxol,
Vincristine, Adriamycin)-induced apoptosis. The PI3K/Akt signaling
pathway has been shown to be involved in cell proliferation and
negatively regulates apoptosis-inducing stimuli. Hence, we examined
if rituximab affects the PI3K/Akt pathway as a mechanism of
chemosensitization of the tumor cells. Ramos B-NHL cells were
treated with predetermined optimal concentration of rituximab (20
ug/ml) for different periods of times (3-20 h) and the cells were
harvested and total cell lysates were prepared. Analysis of lysates
by Western revealed that treatment with rituximab inhibits the
constitutively activated phospho-PI3K, phospho-Akt, phospho-Bad,
(no inhibition of non-phospho related proteins), and downregulated
Bcl-.sub.XL expression as early as 6 h post treatment. Bad and form
complexes and following phosphorylation of Bad by the Akt pathway,
Bcl-.sub.XL dissociates from the complex and participates in the
regulation of resistance to apoptosis. Rituximab treatment of Ramos
cells was shown to inhibit phospho-Bad and there was significant
enhancement of the Bcl-.sub.XL complexed with Bad, thus reducing
free Bcl-.sub.XL and contributing to chemosensitization-mediated by
rituximab. It has been reported that the constitutive activation of
Akt is regulated in large part by PTEN activity. PTEN is a tumor
suppressor that serves as a major negative regulator of the
survival signaling mediated by the PI3K/Akt/protein kinase B
pathways. The constitutive activation of Akt in Ramos was
paralleled by low PTEN levels expression in the cells. Treatment of
Ramos with rituximab resulted in a significant induction of PTEN
expression in the cells. Time kinetic analysis revealed that
rituximab augments PTEN expression as soon as 9 h after treatment
(suggesting a post Akt-induced inhibitor). We then examined the
role of the PI3K/Akt pathway in the regulation of Ramos resistance
to drugs. Treatment of the Ramos cells with the specific Akt
inhibitor LY294002 sensitized the cells to CDDP-induced apoptosis
concomitant with downregulation of Bcl-.sub.XL expression. These
findings demonstrate for the first time that rituximab treatment
can inhibit the constitutive Akt pathway in B-NHL cells and in
addition results in the induction of the expression of the tumor
suppressor PTEN. These findings also reveal different targets whose
modulation can reverse the resistance of tumor cells to
drug-induced apoptosis.
Example 8
Characteristics of Rituximab-resistant B-NHL Clones: Deficiencies
in Rituximab-mediated Changes in Lipid Raft Microdomains and Cell
Signaling
[0279] The chimeric mouse anti-human CD20 mAb Rituximab, alone or
combined with chemotherapy, has significant anti-lymphoma activity.
A subset of patients does not respond initially or following
treatment with rituximab. In order to investigate the mechanism of
rituximab resistance, we have developed rituximab-resistant (RR)
clones of Ramos and Daudi cells following culture with increasing
concentrations of rituximab and limiting dilution analysis of
single cells. The CD20+ expressing RR clones failed to respond to
rituximab-mediated CDC, cytostasis, chemo-sensitization,
cross-linked rituximab-mediated apoptosis (Jazirehi and Bonavida,
Oncogene, 24:2121-43 (2005)). Studies were undertaken to examine
the underlying mechanism of rituximab resistance in the clones. The
RR clones showed over-expression of the complement inhibitor CD59.
In the wild type cells, treatment with rituximab resulted in a
rapid and transient increase in acid-sphingomyelinase activity
concomitant with cellular ceramide generation in lipid rafts.
Rituximab treated cells externalized both acid sphingomyelinase and
ceramide which co-localized with the CD20 receptor (Bezombes et
al., Blood, 104:1166-73 (2004)). Preliminary results show that
rituximab-induced acid sphingomyelinase translocation and ceramide
generation at the cell surface is reduced in RR clones compared to
parental clones. We further analyzed possible mutations in the CD20
coding sequence. There was no difference in mutations between the
parental and RR clones. Since lipid rafts serve as signaling
platforms, thus, it seems that resistance to rituximab in these
clones is due to a defect of the sphingomyelin-ceramide pathway. RR
clones exhibit hyperactivation of the ERK1/2 and NF-kB survival
signaling pathways concomitant with overexpression of Bcl-.sub.XL,
Bcl-2, and Mcl-1. Treatment of RR clones with specific
pharmacological inhibitors of these pathways sensitized the RR
clones to various chemotherapeutic drugs (e.g., CDDP, Vincristine,
Adrimycin, Taxol, Etoposide) and significant synergy in apoptosis
was achieved. These findings demonstrate that the development of
resistance to rituximab treatment may result from alteration of the
cell signaling mediated by rituximab and failure to mobilize the
lipid rafts on the cell membrane and the failure to inhibit
downstream survival signaling pathways. In addition, the findings
suggest the potential therapeutic application of combining
sensitizing agents and conventional therapeutic drugs in the
treatment of rituximab and drug-resistant B-NHL.
Example 9
Development of Rituximab-resistant Lymphoma Clones with Altered
Cell Signaling and Cross-resistance to Chemotherapy: Circumvention
of Acquired Resistance by Specific Pharmacological Inhibitors
(Bortezomib, DHMEQ, PD098059
[0280] The B-cell specific surface marker CD20 (Tedder, T. et al.,
Immunol. Today, 15:450-4 (1994)) does not circulate in the plasma
as a free protein, which could potentially block Ab binding to the
cells (Andeson, K. et al., Blood, 63:1424-33 (1984)). Also, it is
neither internalized upon Ab ligation (Press, 0. et al., Blood,
69:584-91 (1987)) nor shed from cell surface (Einfeld, D. et al.,
EMBO J, 7:711-7 (1988)); properties that make CD20 an ideal target
for immunotherapy of NHL. The chimeric mouse anti-human CD20 mAb
rituximab (IgG1.kappa.) binds with high affinity to CD20 expressing
cells. It is the first FDA approved mAb for NHL treatment
(Grillo-Lopez, A., Int J Hematol., 76:385-93 (2002)).
[0281] Rituximab has been an important addition to the therapeutic
armamentarium against low-grade follicular NHL (Dillman, R., Semin
Oncol., 30:434-47 (2003)). Furthermore, its utilization alone or
combined with chemotherapy is considered as first line therapeutic
option for other types of hematological malignancies (Lin, T. et
al., Semin Oncol., 30:483-92 (2003); Hiddemann, W. et al., Semin
Oncol., 30:16-20 (2003); Ghobrial, I. et al., Lancet Oncol.,
4:679-85 (2003)) improving patients' survival. Its usage is also
extended to other pathologic states culminating in long-lasting
response (Risken, N. et al., Neth J Med., 61:262-5 (1993); Pels, H.
et al., Onkologie, 26:351-4; Reams, B. et al., Chest, 124:1242-9
(2003); Weichert, Z. et al., J Cutan Med. Surg., 7:460-3 (2003)).
Rituximab exerts significant anti-tumor activity in vivo (Reff, M.
et al., Blood, 83:435-5 (1994)) via inhibition of cell
proliferation or triggering multiple cell-damaging mechanisms
including antibody-dependent cellular cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC) and apoptosis (Maloney, D.,
J Clin Oncol., 23:6421-8 (2005); Smith, M., Oncogene, 22:7359-68
(2003)). It also augments the cytotoxic effects of drugs on
drug-resistant NHL B-cells (Jazirehi, A. et al., Oncogene,
24:2121-43 (2005)). Nonetheless, the contribution of these
mechanisms on primary normal and malignant B-cells in vivo and the
molecular mechanisms of rituximab action need to be defined.
[0282] We have explored the effects of modifications of signaling
pathways by rituximab on its chemo-sensitizing attributes. To this
end we have recently reported that rituximab, via inhibition of
NF-.kappa.B and ERK1/2MAPK pathways, reduces Bcl-.sub.XL expression
and chemo-sensitizes NHL B-cells (Jazirehi, A. et al., Cancer
Research, 64:7117-26 (2004); Jazirehi, A. et al., Cancer Research,
65:264-76 (2005)). Activation of NF-.kappa.B and ERK1/2 pathways
are emerging as major mechanisms of tumor cell drug-resistance and
induce their rapid proliferation. Thus, interruption of these
pathways is a target for therapeutic intervention and may confer
drug-sensitivity (Ghosh, S. et al., Cell, 109:S81-S96 (2002); Dent,
P. et al., Clin Cancer Res, 7:775-83 (2001)), which has proven
successful in enhancing the apoptotic effects of TNF-.alpha. and
CPT-11 resulting in tumor regression in vivo (Wang, C. et al.,
Nature Medicine, 5:412-7 (1999)). Inhibition of NF-.kappa.B and
ERK1/2 pathways was shown by decrease in phosphorylation and kinase
activities of the signaling molecules and reduced NF-.kappa.B and
AP-1 DNA-binding ability (DBA) concomitant with reduction in the
expression of their common down-stream target (24) Bcl-.sub.XL.
[0283] The superior efficacy of CHOP+rituximab (R-CHOP) compared to
CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone)
alone in elderly DLBCL patients was reported, where the combination
therapy resulted in higher rates of complete remission and survival
(Coiffier, B. et al., Anticancer Drugs, 2:S43-50 (2002)) and also
increases overall survival in aggressive Bcl-2-positive and
-negative patients (Mounier, N. et al., Haematologica, 91:715-6
(2006)). Despite its well-established clinical efficacy, a
sub-population of patients, via an elusive mechanism, does not
respond to rituximab and/or acquires resistance upon long-term
rituximab therapy (Maloney, D., J Clin Oncol., 23:6421-8 (2005);
Smith, M., Oncogene, 22:7359-68 (2003)). Based on previous reports
we hypothesized that development of rituximab-resistance may be
related to tumor cells' failure to respond to rituximab-mediated
signaling. Further, the unresponsiveness of the cells to drug
therapy (alone or combined with rituximab) may be due to
hyper-activation of survival signaling pathways and up-regulation
of resistant-factors. Due to challenges in obtaining
patient-derived specimens for analysis, and to recapitulate various
aspects of acquired rituximab-resistant situations,
rituximab-refractory (RR) clones were generated (Jazirehi, A. et
al., Blood, 104:3410 (2004))*. Using a battery of functional and
biochemical assays, representative clones were compared to parental
cells to examine alterations in rituximab-mediated effects to
examine the above hypotheses. Different RR clones have also been
established and analyzed (Olejniczak, S. et al., Blood, 106:4819
(2005))**. The following objectives were investigated: 1)
phenotypic and functional properties of RR clones (e.g.,
differences regarding CD20 surface expression, proliferation, CDC,
cross-linked rituximab-mediated apoptosis), 2) chemo-sensitivity of
the clones and chemo-sensitization by rituximab, 3) activation
status of ERK1/2 and NF-.kappa.B pathways, 4) expression of Bcl-2
family members, and 5) effects of various inhibitors of survival
pathways on reversal of chemo-resistance.
Materials and Methods
Cell Lines and Clones.
[0284] C.D20.sup.+ human Burkitt's lymphoma B-cell lines Daudi and
Ramos were obtained from ATCC (Bethesda, Md.). For the generation
of RR clones wt cells were grown in the presence of step-wise
increasing concentrations of rituximab (5-20 .mu.g/ml-10 weeks).
Single cells were then subjected to three consecutive rounds of
limiting dilution analysis (LDA) (Jazirehi, A. et al., Blood,
104:3410 (2004)). Single cells were propagated and maintained in
RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal
bovine serum (FBS) (Jazirehi, A. et al., Mol. Cancer Therapeutics,
2:1183-93 (2003)). Clones were supplemented with rituximab (20
.mu.g/ml) once a week and grown in rituximab-free medium at least
one week prior to analysis. Cultures were incubated in controlled
atmosphere incubator at 37.degree. C. with saturated humidity at
0.5.times.10.sup.6 cells/ml.
[0285] Reagents.
[0286] Paclitaxel, CDDP, VP-16, ADR, and vincristine were purchased
from Sigma (St. Louis, Mo.) and were diluted in DMSO. DMSO
concentration did not exceed 0.1 % in any experiment. Mouse
anti-Bcl-.sub.XL, -Mcl-1,-Bcl-2 mAbs were purchased from Santa Cruz
Biotechnology (Santa Cruz, Calif.) and DAKO (Carpinteria, Calif.),
respectively. Mouse anti-p-I.kappa.B-.alpha. (Ser.sup.32/36),
-actin mAbs were obtained from Imgenex (San Diego, Calif.) and
Chemicon (Temeculla, Calif.), respectively. Rabbit
p-IKK.alpha./.beta. [Ser.sup.180/181] Ab was obtained from Cell
Signaling (Beverley, Mass.). Rabbit anti-p-ERK1/2
(Thr.sup.185/Tyr.sup.187) Ab, MAPK kinase substrate-4 (aa 172-192)
and PD098059 were obtained from Biosource (Camarillo, Calif.).
2MAM-A3 was purchased from Biomol (Plymouth, Pa.). DHMEQ was
provided by Dr. K. Umezawa (Tokyo, Japan). Rituximab and bortezomib
were procured commercially.
[0287] Surface CD20 Expression.
[0288] Cells (2.times.10.sup.6) were washed twice with ice cold
1.times.PBS and stained with 1 .quadrature.g mouse anti-human CD20
mAb (IDEC-2B8; IDEC Pharmaceuticals, San Diego, Calif.) or isotype
control (pure IgG1) (20 minutes on ice, light protected). Then, the
cells were washed twice with ice cold 1.times.PBS, stained with
FITC-labeled secondary Ab (30 minutes on ice, light protected) and
subjected to FACS analysis.
[0289] Immunoblot Analysis.
[0290] Cells (10.sup.7) were either grown in complete medium or
complete medium supplemented with various inhibitors. Cells were
lysed at 4.degree. C. in radioimmuno-precipitation assay (RIPA)
buffer [50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium
deoxycholate, 150 mM NaCl] supplemented with one tablet of protease
inhibitor cocktail (Complete Mini; Roche). A detergent-compatible
protein (DC) assay kit (Bio-Rad, Hercules, Calif.) was used to
determine protein concentration. An aliquot of total protein lysate
was diluted in an equal volume of 2.times.SDS sample buffer, boiled
for 10 min, and cell lysates were electrophoresed on 12% SDS-PAGE
gels. Western blot was carried out as described (Jazirehi, A. et
al., Mol. Cancer Therapeutics, 2:1183-93 (2003)). The relative
intensity of bands, hence, relative alterations in protein
expression, was assessed by densitometric analysis of digitized
images using public domain NIH image program
(http://rsb.info.nih.gov/nih-image/).
[0291] Assessment of Apoptosis. A. DNA Fragmentation Assay.
[0292] Percentage of apoptotic cells was determined by evaluation
of propidium iodide (PI) stained preparations of cells using an
Epic.sub.XL flow-cytometer. Cellular debris was excluded from
analysis by raising the forward scatter threshold, and DNA content
of the intact nuclei was recorded on a logarithmic scale
(Nicoletti, I. et al., J Immunol. Methods, 139:271-9 (1991)).
Percent apoptosis is represented as percentage of hypodiploid cells
accumulated at sub-G0 phase of cell cycle.
[0293] Evaluation of Active Caspase-3 Levels.
[0294] Levels of active caspase-3 were evaluated with FITC-labeled
anti-active caspase-3 mAb (PharMingen, San Diego, Calif.)
(Jazirehi, A. et al., Mol. Cancer Therapeutics, 2:1183-93
(2003)).
Assessment of Viable Cell Recovery.
[0295] This was assessed using standard XTT assay kit (Roche,
Indianapolis, Ind.) that measures metabolic activity of viable
cells (Scudiero, D. et al., Cancer Res., 48:4827-33 (1998)).
Percent cell recovery was calculated using background-corrected
reading as follows: % Cell Recovery=[(OD of sample wells/OD of
untreated cells)].times.100.
Electrophoretic Mobility Shift Analysis.
[0296] The DBAs were evaluated using biotin-labeled oligonucleotide
AP-1 (5'-CGCTTGATGACTCAGCCGGAA-3') (Lee, W et al., Cell, 49:741-52
(1987)) and NF-.kappa.B (5'-AGTTGAGGGGACTT TCCCAGGC-3') probes
(Harada, H. et al., Mol. Cell Biol., 14:1500-9 (1994)) using EMSA
kit (Panomics, Inc., Redwood City, Calif.) according to
manufacturer's instructions. 10 .mu.g of nuclear extracts were
subjected to denaturing 5% PAGE and developed (Jazirehi, A. et al.,
Cancer Research, 64:7117-26 (2004); Jazirehi, A. et al., Cancer
Research, 65:264-76 (2005)).
Immune-complex Kinase Assay.
[0297] The kinase activity of IKK and MEK1/2 was assessed by their
ability to phosphorylate I.kappa.B-.alpha. (Ser.sup.32/36) and MAPK
kinase substrate 4 (Thr.sup.185/Tyr187) using a slightly modified
version of previous methods (Alessi, D. et al., J. Biol. Chem.,
270:27489-94 (1995); Jazirehi, A. et al., Cancer Research,
64:7117-26 (2004); Jazirehi, A. et al., Cancer Research, 65:264-76
(2005)).
Quantitative Real-time PCR (qPCR).
[0298] Samples were analyzed in triplicate with iQ SYBR Green
Supermix using iCycler Sequence Detection System (BioRad). Total
RNA was extracted from 10.sup.7 cells for each condition with 1
ml/sample of STAT-60 reagent and quantified by 3.1.2 NanoDrop
ND-1000 spectrophotometer. 3 .mu.g of total RNA was reversed to
first-stranded cDNA for 1 h at 42.degree. C. with 200 units
SuperScript II RT and 20 .mu.M random hexamer primers.
Amplification of 2.5 .mu.l of cDNAs was performed using
gene-specific primers. Internal control for equal cDNA loading in
each reaction was assessed using G-3-PDH primers. Amplicons were
resolved by 2% gels for confirmation and were of expected size.
Percentages of expression of each molecule were calculated with the
assumption that control samples were considered as 100%.
[0299] Statistical Analysis.
[0300] Assays were set up in triplicates and results were expressed
as mean.+-.standard deviation (STD). Statistical analysis and P
values were calculated by two-tailed paired t test with a
confidence interval (CI) of 95% for determination of significance
of differences between treatment groups. (P<0.05: significant).
ANOVA was used to test significance among the groups using InStat
2.01 software.
Example 9A
Phenotypic and Functional Properties of the RR Clones
(i) Diminished Surface CD20 Expression and Failure to Respond to
Rituximab-Mediated Inhibition of Cell Growth and Apoptosis
Following Cross-linking.
[0301] Wild type (wt) cells and Ramos-RR1 and Daudi-RR1 clones were
stained with isotype control (pure IgG1; gray lines) or
FITC-labeled anti-CD20 mAb (IgG1 subtype; solid black lines) and
subjected to FACS analysis. As measured by mean fluorescence
intensity (MFI), wt cells show significant CD20 surface expression
while the clones exhibit .about.40-50% reduction in surface CD20
(Ramos: 468.4.+-.14.0 vs. 264.+-.9.6, Daudi: 346.+-.11.4 vs.
156.4.+-.10.2) (FIG. 6A). Similar results were obtained in multiple
independent experiments with other Ramos-RR and DLBCL-RR clones
(data not shown) suggesting that continuous rituximab treatment
results in diminished, but not complete loss of, surface CD20
expression on RR clones.
[0302] To assess the ability of RR clones to respond to growth
inhibitory effects of rituximab, both the wt and the clones were
left either untreated or treated with a predetermined concentration
of rituximab for wt cells (20 .mu.g/ml-24 h) (34). An aliquot
(10.sup.4) of cells was used in a standard XTT viability assay.
Rituximab exerts an inhibitory effect on the wt cells (Ramos: 27%,
Daudi: 46%) while it fails to reduce the growth of RR clones
(Ramos-RR1: 98%, Daudi-RR1: 106%) (FIG. 6B). Higher concentrations
(50, 100 .mu.g/ml) or longer exposure time (up to 48 h) of
rituximab did not alter the phenotype of the clones (data not
shown); thus, 20 .mu.g/ml rituximab (24 h) was used in subsequent
studies. Results of the XTT assay were confirmed by trypan blue dye
exclusion and FACS (data not shown), which essentially showed that
rituximab fails to induce apoptosis or inhibit cell growth in RR
clones. Untreated RR clones exhibited homotypic aggregation that
remained unaffected by rituximab. In contrast, wt cells grew as
suspended non-aggregated and rituximab caused them to form clumps
(FIG. 6C). These results indicate that while rituximab efficiently
lowers the growth rate of the wt cells, it fails to reduce the
growth of RR clones.
[0303] Next, we assessed the ability of cross-linked rituximab to
induce apoptosis in RR clones. The wt cells and the clones were
pre-treated with optimal concentration of cross-linked rituximab
(50 .mu.g/ml anti-human immunoglobulin (hIg)+20 .mu.g/ml
rituximab-24 h) (Shan, D. et al., Blood, 91:1644-52 (1998)) and
subjected to apoptosis assay. DNA fragmentation assay for apoptosis
detection in subsequent experiments was confirmed by measuring
active caspase-3 levels (28, data not shown). Neither rituximab
(Ramos-RR1: 11.0.+-.0.8%, Daudi-RR1: 6.7.+-.0.6%) nor the anti-hIg
(Ramos-RR1: 10.2.+-.1.1%, Daudi-RR1: 12.8.+-.0.5%) alone
efficiently killed the cells. However, combination of the two
agents (cross-linked rituximab) induced significant levels of
apoptosis in Ramos (29.2.+-.2.4%) and Daudi (32.8.+-.2.1%) wt
cells, while cross-linked rituximab moderately killed RR clones
(FIG. 6D) suggesting that clones have developed higher threshold
(Ramos-RR1: 2.86, Daudi-RR1: 2.56 folds) and, unlike the wt cells,
do not efficiently respond to cross-linked rituximab-mediated
apoptosis.
(ii) Failure to Respond to CDC
[0304] The ability of rituximab to mediate CDC in RR clones
compared to the wt cells was assessed by analyzing the percentage
of dead cells that were treated with human AB serum as source of
complement (5, 10%-24 h). As shown in FIG. 6E, wt cells exhibited
modest sensitivity to the cytotoxic effects of AB serum (as a
function of serum concentration) (Ramos: 15.5.+-.1.3%, Daudi:
14.3.+-.0.9%); an effect that was significantly augmented in the
presence of rituximab (Ramos: 36.9.+-.1.5% vs. 11.0.+-.0.8%, Daudi:
29.2.+-.1.6% vs. 6.2.+-.0.6%). There was augmentation of
cytotoxicity with 10% serum than with 5%. However, compared to wt
cells, the clones were less sensitive to human AB serum, and
rituximab failed to enhance their sensitivity (Ramos-RR1:
14.2.+-.1.6% vs. 7.8.+-.0.6%, Daudi-RR1: 11.6.+-.2.4% vs.
6.6.+-.1.1%). Increasing serum concentration (15%) neither enhanced
their sensitivity nor augmented rituximab-mediated CDC of the
clones (data not shown). These results show that wt cells are
sensitive to CDC which is enhanced by both serum levels and
rituximab treatment, while long-term rituximab exposure is
accompanied by higher CDC-resistance in the clones (Ramos-RR1: 2.6
fold, Daudi-RR1: 2.5 folds) and rituximab fails to augment CDC.
(iii) Failure of Rituximab to Chemo-sensitize the RR Clones
[0305] Augmentation of the cytotoxic effects of drugs is an
established property of rituximab (Jazirehi, A. et al., Oncogene,
24:2121-43 (2005)). To assess the ability of rituximab to
chemo-sensitize RR clones, wt cells and the clones were pretreated
with rituximab, subsequently treated with various concentrations of
paclitaxel (0.1-10 nM) and subjected to apoptosis assay. Paclitaxel
was used as a representative drug; similar results were obtained
with etoposide (VP-1 6) and cis-platinum (CDDP) (data not shown).
Rituximab significantly augmented the apoptotic effect of
paclitaxel in wt Ramos and Daudi cells in a concentration-dependent
manner (range 45-58% apoptosis) (FIG. 6F). However, RR clones had
higher resistance to paclitaxel, and rituximab was incapable of
augmenting paclitaxel-induced apoptosis (FIG. 6F). 2.5-5 folds
higher concentrations of rituximab failed to sensitize the clones
(data not shown). These results suggest that while rituximab
efficiently chemo-sensitizes wt cells it is incapable of
chemo-sensitizing the clones suggestive of higher resistance of RR
clones to rituximab-mediated chemo-sensitization.
Example 9B
Development of Higher Drug-resistance in RR Clones
[0306] Since RR clones were not chemo-sensitized by rituximab,
their sensitivity against a battery of drugs including paclitaxel,
vincristine, VP-16, adriamycin (ADR), and CDDP was examined.
Compared to wt cells, which exhibit moderate sensitivity to these
drugs in a concentration-dependent manner, clones exhibited higher
apoptosis threshold to these drugs, albeit to varying degrees. As
such, Ramos-RR1 showed 1.54 (136%), 1.42 (130%), 2.3 (158%), 1.8
(145%), 1.41 (120%) folds and Daudi-RR1 showed 1.63 (139%), 1.66
(140%), 1.94 (149%), 2.97 (167%), 1.95 (149%) folds resistance to
paclitaxel, vincristine, ADR, VP-16, and CDDP, respectively,
compared with their respective wt cells (FIG. 7A). Prolonged
incubation time (48 h) did not significantly augment drug cytotoxic
in the clones (data not shown) suggesting that compared to wt
cells, RR clones exhibit higher (1.41-2.97 fold) drug-resistance.
Functional analysis of the multi-drug resistance (MDR) pump showed
the existence of functional MDR pump in both wt cells and the
clones. Further, RR clones exhibited no functional impairment of
MDR pump (.+-.rituximab, data not shown) suggesting that higher
drug-resistance in the clones is independent of the MDR pump.
Example 9C
Over-expression of Bcl-2, Bcl-.sub.XL and Mcl-1 in RR clones.
[0307] RR clones did not respond to rituximab-mediated
chemo-sensitization and exhibited higher drug-resistance. Previous
findings have established Bcl-.sub.XL as an important
resistant-factor (Jazirehi, A. et al., Cancer Research, 64:7117-26
(2004); Jazirehi, A. et al., Cancer Research, 65:264-76 (2005));
thus, we evaluated Bcl-.sub.XL levels and other anti-apoptotic
Bcl-2 family members in the clones. Total RNA was extracted and
converted to first stranded cDNA which was subjected to real-time
quantitative-PCR (qPCR) analysis. RR clones exhibited increased
expression of Bcl-2 (Ramos-RR1: 3.6, Daudi-RR1: 3.2 fold),
Bcl-.sub.XL (Ramos-RR1: 8.2, Daudi-RR1: 4.2 fold), Mcl-1
(Ramos-RR1: 3.4, Daudi-RR1: 2.8 fold) at the transcription level
(FIG. 7B). Immunoblot showed that clones exhibit higher Bcl-2,
Bcl-.sub.XL, Mcl-1 protein levels (.about.2.3-5.0 fold) compared to
wt cells. Interestingly, expression levels of these proteins
remained unaffected by rituximab treatment of the clones, while
Bcl-.sub.XL levels in wt cells were reduced (FIG. 7C). Notably,
expression levels of other pro- and anti-apoptotic factors
(Bcl-.sub.xS, Bfl-1, /A1, Bad, Bax, Bid, Bak, c-IAP-1,-2, survivin,
XIAP) were similar in wt cells and RR clones (.+-.rituximab) (data
not shown). These results show that clones express higher levels of
protective factors which may explain their unresponsiveness to
rituximab-mediated chemo-sensitization and higher drug-resistance.
Also rituximab was unable to reduce the levels of resistant-factors
suggesting that signaling pathways in the clones are no longer
responsive to rituximab.
Example 9D
Hyper-activation of the ERK1/2 and NF-.quadrature.B Signaling
Pathways in the RR Clones
[0308] The above findings suggest that the dynamics of the cellular
signaling pathways are altered in the clones. Rituximab inhibits
ERK1/2 and NF-.kappa.B pathways leading to reduced DBA of AP-1 and
NF-.kappa.B transcription factors in wt cells (Jazirehi, A. et al.,
Cancer Research, 64:7117-26 (2004); Jazirehi, A. et al., Cancer
Research, 65:264-76 (2005)). Thus, we examined the activation
status of these pathways in the clones. Whole cell extracts of wt
cells and the clones (.+-.rituximab) were subjected to immunoblot
for components of NF-.kappa.B and ERK1/2 pathways. The
phosphorylation-dependent state of IKK, I.kappa.B-.alpha. and
ERK1/2 was higher in the clones (.about.3.2-4.8 folds) than wt
cells. Basal levels of these signaling molecules remained
unaffected in wt and RR clones (.+-.rituximab, data not shown).
Rituximab significantly reduces the phosphorylation of these
molecules in wt cells (Jazirehi, A. et al., Cancer Research,
64:7117-26 (2004); Jazirehi, A. et al., Cancer Research, 65:264-76
(2005)), an effect that is not observed in clones (FIG. 8A)
suggesting that molecular switches responsible for
rituximab-mediated de-phosphorylation of these molecules are no
longer operative in clones.
[0309] To ascertain the observed hyper-phosphorylation results in
increased activity of NF-.kappa.B and ERK1/2 pathways, immune
complex kinase assays were performed to assess IKK and MEK1/2
kinase activities of the clones (.+-.rituximab) using
I.kappa.B-.alpha. peptide (IKK substrate) and MAPK kinase
substrate-4 (ERK1/2 substrate). Untreated clones showed
significantly increased kinase activities as shown by increased
ability of the lysates to phosphorylate their specific substrates,
whereby rituximab did not reduce the IKK and MEK1/2 kinase
activities. This phenomenon was not observed by I.kappa.B-.alpha.
peptide S32/36A (data not shown). In contrast, in wt cells
rituximab diminishes the kinase activity of IKK and MEK1/2 (FIG.
8B), thus, increased phosphorylation of signaling molecules
culminates in higher kinase activity of these pathways in the
clones. Next, alterations in the DBA of NF-.kappa.B and AP-1 in the
clones were examined. Biotin-labeled oligonucleotides probes
comprising the NF-.kappa.B (Harada, H. et al., Mol. Cell Biol.,
14:1500-9 (1994)) and AP-1 (Lee, W et al., Cell, 49:741-52 (1987))
consensus binding sites were used in EMSAs which reveal that
compared to wt cells NF-.kappa.B and AP-1 DBAs are increased in the
clones and rituximab fails to reduce their DBA. Rituximab-induced
decrease in NF-.kappa.B and AP-1 DBA was only observed in wt cells.
Specificity of EMSA was corroborated using appropriate controls
(FIG. 8C). Specific inhibitors (DHMEQ, and PD098059) preferentially
reduced NF-.kappa.B and AP-1 DBA. Collectively, these results show
that NF-.kappa.B and ERK1/2 pathways are constitutively
hyper-activated in RR clones and denote the inability of rituximab
to negatively regulate the activities of these pathways in the
clones unlike wt cells. Hyper-activation of these pathways will
lead to enhanced transcription of their respective anti-apoptotic
target genes leading to higher drug-resistance of the clones.
Example 9E
Chemo-sensitization of RR Clones by Pharmacological Inhibitors of
ERK1/2 and NF-.kappa.B Pathways
[0310] The NF-.kappa.B and ERK1/2 pathways are hyper-activated in
RR clones leading to over-expression of Bcl-2, Bcl-.sub.XL, Mcl-1
all of which are unaffected by rituximab, prompting us to
investigate whether inhibition of these pathways or Bcl-2 members
can reverse chemo-resistance. Since these pathways have higher
activities in RR clones, higher concentrations of inhibitors were
required for chemo-sensitization of the clones than those used for
wt cells; non-toxic effective concentrations of which were
determined by pilot studies (data not shown). Cells were left
either untreated or pre-treated with DHMEQ (36), bortezomib (Goy,
A. et al., Clin. Lymphoma, 4:230-7 (2004)) and PD098059 (Alessi, D.
et al., J Biol. Chem., 270:27489-94 (1995)). Escalating
concentrations of various drugs were then added and the percentage
of apoptosis was measured. In Ramos-RR1, while DHMEQ induces
10.1.+-.2.1% apoptosis, it significantly augments the apoptotic
effects of drugs (paclitaxel: 20.4.+-.3.0%.fwdarw.46.5.+-.2.3%,
ADR: 14.3.+-.3.3%.fwdarw.32.4.+-.2.9%, VP-16:
10.3.+-.1.1%.fwdarw.53.0.+-.2.6%, CDDP:
19.0.+-.0.7%.fwdarw.61.2.+-.2.1%, vincristine:
15.3.+-.1.1%.fwdarw.53.5.+-.2.2%). In Daudi-RR1 DHMEQ induces
4.9.+-.1.6% apoptosis and augments drug efficacy (paclitaxel:
14.9.+-.3.2%.fwdarw.42.0.+-.1.9%, ADR:
12.4.+-.2.5%.fwdarw.40.5.+-.3.1%, VP-16:
11.8.+-.0.9%.fwdarw.32.0.+-.2.2%, CDDP:
25.7.+-.3.1%.fwdarw.36.6.+-.0.9%, vincristine:
14.2.+-.1.7%.fwdarw.36.6.+-.2.3%) (FIG. 9A-C). Similar patterns of
significant dose-dependent chemo-sensitization of RR clones were
observed, though for simplicity only the values pertaining to the
highest drug concentration are presented (FIG. 12, Table 1A).
Enhanced cytotoxicity by DHMEQ was 2.3-5.1 and 1.4-3.3 folds, by
bortezomib was 1.8-5.1 and 1.7-3.3 folds and by PD098059 was
1.8-3.1 and 1.5-2.7 folds in Ramos-RR1 and Daudi-RR1, respectively
(FIG. 12, Table 1B). The ability of inhibitors to significantly
chemo-sensitize the RR clones (1.5-5.1 folds), indicates that
inhibition of NF-.kappa.B and ERK1/2 pathways can avert
chemo-/rituximab-resistance in clones to sub-toxic drug
concentrations.
[0311] Since inhibitors efficiently chemo-sensitized the clones, we
assessed their effect on expression of the resistant-factors. As
depicted by qPCR inhibitors reduced mRNA levels of Bcl-2,
Bcl-.sub.XL and Mcl-1 by 1.2-4.8 folds and 1.2-6.6 folds (FIG.
10A), and immunoblot showed 1.25-3.3 folds and 1.1-3.3 folds
decrease in their protein levels in Ramos-RR1 and Daudi-RR1,
respectively (FIG. 10B) further showing the involvement of
NF-.kappa.B and ERK1/2 pathways in the expression of
resistant-factors.
[0312] The chemo-protective role of the over-expressed Bcl-2,
Bcl-.sub.XL and Mcl-1 in clones was further confirmed by
pre-treatment with 2MAM-A3 (Tzung, S-P et al., Nature Cell Biology,
3:183-91 (2001)) which chemo-sensitized them at levels comparable
with those achieved by rituximab. In wt Ramos 2MAM-A3 augmented
paclitaxel cytotoxicity by 1.96 folds
(13.6.+-.1.3%.fwdarw.26.7.+-.2.2%), which was 4.71 folds
(8.42.+-.2.1%.fwdarw.39.7.+-.2.4%) in Ramos-RR1. Similar pattern
was observed in Daudi (wt: 1.98 fold, RR1: 3.2 folds) (FIG. 10C).
These findings support our contention that over-expression of
anti-apoptotic Bcl-2 members upon prolonged rituximab treatment
protects the cells against drug-induced apoptosis and their
functional impairment is critical for chemo-sensitization.
[0313] These B-NHL RR clones which exhibit a different phenotypic
profile compared to wt cells. Using various biochemical and
functional assays, compared to wt cells, RR clones express lower
levels of surface CD20, do not respond to either growth inhibition
by rituximab, rituximab-mediated CDC or cross-linked
rituximab-induced apoptosis. Further, RR clones are not
chemo-sensitized by rituximab and exhibit higher drug-resistance
(1.41-2.97 fold). Two major survival pathways (NF-.kappa.B and
ERK1/2) are constitutively hyper-activated in the clones leading to
over-expression of resistant-factors (Bcl-2, Bcl-.sub.XL , Mcl-1)
(.about.2.3-5.0 fold). Pharmacological inhibition of Bcl-2 family
members (by 2MAM-A3), NF-KB (by DHMEQ, bortezomib), and ERK1/2
pathways (by PD098059) averts the drug-resistant phenotype and the
clones undergo apoptosis in response to low concentrations of
various drugs (FIG. 11).
[0314] Reducing the proliferation rate of tumor cells is postulated
as one of rituximab's potential modes of action (Maloney, D., J
Clin Oncol., 23:6421-8 (2005); Smith, M., Oncogene, 22:7359-68
(2003)). Rituximab treatment of wt cells reduces their growth rate
(Jazirehi, A. et al., Mol. Cancer Therapeutics, 2:1183-93 (2003)),
though, the clones grew at similar rates as wt cells and rituximab
was incapable of inhibiting their growth (FIG. 6B) consistent with
the higher growth rate and progressive nature of relapsed
lymphomas, suggesting that RR clones have lost the ability to
undergo rituximab-mediated growth-reduction possibly through a
defective ceramide (CER)- acid sphingomyelinase (A-SMase) pathway
(Bezombes, C. et al., Blood, 104:1166-73 (2004)). Compared to wt
cells, RR clones exhibit higher resistance to CDC. Rituximab
pretreatment significantly enhanced CDC in wt cells; an effect that
was not noticed in the clones. Increasing serum concentrations
enhanced rituximab-mediated CDC in wt cells but not in clones (FIG.
6D) consistent with our preliminary findings showing increased
expression of complement inhibitors on the clones (data not shown).
Further studies are warranted to delineate the role of complement
inhibitors in CDC-resistance of the RR clones. Cross-linked
rituximab induced significant apoptosis (FIG. 6E) and cytostasis
(data not shown) on wt cells. Neither induction of apoptosis nor
growth inhibition was observed on treatment of the clones with
cross-linked rituximab, suggesting that RR clones have developed
higher threshold in response to biological effects of cross-linked
rituximab. The above effects were independent of rituximab
concentration as higher (2.5-5 fold) concentrations of rituximab
failed to reverse the phenotype of the clones (data not shown).
[0315] Rituximab binds to B-cell restricted cell surface CD20,
thus, exerting its effects. Various mechanisms are postulated for
rituximab-resistance including transient CD20 down-regulation
(Kennedy, A. et al., J Immunol., 172:3280-8 (2004)), loss of CD20
(Haidar, J. et al., Eur J Haematol, 70:330-2 (2003)) and
circulating CD20 (Manshouri, T. et al., Blood, 101:2507-13 (2003)).
Substantial CD20 surface expression was detected on wt cells,
still, RR clones exhibited about 50% reduction in surface CD20
expression (FIG. 6A). The significance of reduced CD20 expression
in clones requires further investigation as several lines of
evidence suggest that the biological effects of rituximab therapy
is autonomous of the intensity of surface CD20 even in vivo
(Kennedy, A. et al., J immunol., 172:3280-8 (2004)). Thus, failure
of rituximab to exert its biological effects on RR clones may be
independent of diminished CD20. It may be due to the activation
status of the clones and/or aberrant cellular signaling as
deregulation of the signaling pathways or aberrant expression of
signaling molecules contribute to acquired chemo-resistance
(Manshouri, T. et al., Blood, 101:2507-13 (2003); Pommier, Y. et
al., Oncogene, 23:2934-49 (2004)). Analysis of the signaling
pathways in the clones revealed hyper-activation of ERK1/2 and
NF-.kappa.B pathways leading to over-expression of their
down-stream resistant- factors Bcl-2, Bcl-.sub.XL and Mcl-1, and
the exhibition of higher drug-resistance (1.41-2.97 fold)
concordant with the protective role of Bcl-2 family members (Wada,
T. et al., Oncogene, 23:2838-49 (2004); Minn, A. et al., Blood,
86:1903-10 (1995)) suggesting that the selective pressure applied
by prolonged rituximab treatment has co-selected for cells that
express higher levels of anti-apoptotic proteins, which have lost
the capacity to undergo apoptosis in response to various stimuli.
The possibility of the pre-existence of resistant cells in the
native culture is not ruled out. In fact, by no criteria thus far
rituximab has altered the biological properties of 100% of the
cells. However, the resistant sub-clones in native culture will
dominate the sensitive population on long-term rituximab treatment
as the sensitive cells will be eliminated over time. However, there
is no unequivocal evidence, as yet, that this is the dominant
mechanism in vivo. Since drugs utilize apoptosis as a mean of
exerting their effects, thus, drug-resistant tumors develop
cross-resistance to apoptosis induced by structurally and
functionally distinct stimuli including immunotherapy and vs. Thus,
as NHL cells develop resistance to rituximab, they may also develop
cross-resistance to drugs and immune system, consistent with our
observation that drug-resistant RR clones also exhibit higher
resistance to tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) and anti-Fas agonistic Ab
(Jazirehi, A. et al., Blood, 106:1514 (2005)). Rituximab
pre-treatment of wt cells, not the clones, efficiently sensitized
them to drugs and TRAIL (Jazirehi, A. et al., Blood, 106:1514
(2005)).
[0316] Unlike wt cells (Jazirehi, A. et al., Cancer Research,
64:7117-26 (2004); Jazirehi, A. et al., Cancer Research, 65:264-76
(2005)), rituximab was incapable of inhibiting hyper-activated
NF-.kappa.B and ERK1/2 pathways in the clones. Thus, constitutive
hyper-activation of these pathways appear to confer higher
drug-resistance (Ghosh, S. et al., Cell, 109:S81-S96 (2002); Dent,
P. et al., Clin Cancer Res, 7:775-83 (2001)), and their inhibition
could potentially avert the chemo-resistance; prompting us to
evaluate the chemo-sensitizing effects of specific inhibitors of
NF-.kappa.B and ERK1/2 pathways as well as bortezomib (Velcade)
(Goy, A. et al., Clin. Lymphoma, 4:230-7 (2004)). DHMEQ is a unique
inhibitor of NF-.kappa.B acting at the level of nuclear
translocation, completely inhibits NF-.kappa.B DBA, inhibits the
growth of human hormone-refractory prostate and bladder cancer
cells and at high concentrations induces apoptosis (Ariga, A. et
al., J. Biol. Chem., 277:24626-30 (2002); Kikuchi, E. et al.,
Cancer Res., 63:107-10 (2003)). PD098059 exerts its effects by
specifically binding to inactive form of MEK1/2 and prevents its
activation by Raf-1, thus, inhibiting ERK1/2 activation (Alessi, D.
et al., J. Biol. Chem., 270:27489-94 (1995)). Bortezomib is
approved for the treatment of multiple myeloma and has significant
single-agent activity against certain subtypes of NHL (O'Connor, O.
et al., J Clin Oncol., 23:676-84 (2005)). These inhibitors
efficiently sensitized the RR clones to structurally and
functionally distinct drugs including topoisomerase II inhibitor,
DNA alkylating agents, and microtubule poisons, albeit to varying
degrees (FIG. 12, Table 1). The inhibitors also reduced Bcl-2,
Bcl-.sub.XL, MCl-1 levels further suggesting that deregulated
signaling culminates in over-expression of anti-apoptotic proteins
in RR clones leading to higher drug-resistance. The
chemo-protective role of Bcl-2 family members was confirmed by
using 2MAM-A3, a specific inhibitor that binds to the hydrophobic
groove formed by the highly conserved BH1, BH2 and BH3 domains,
thus, impairing the function of Bcl-2, Bcl-.sub.XL and Mcl-1
(Tzung, S-P et al., Nature Cell Biology, 3:183-91 (2001)). 2MAM-A3
efficiently chemo-sensitized the clones, further attesting that
higher expression of resistant-factors protects RR clones from
drug-induced apoptosis. Hence, aberrations in the normal dynamics
of cellular survival pathways upon continuous rituximab exposure
contribute to acquired rituximab- and/or drug-resistance while
interruption of these pathways leads to chemo-sensitization of RR
clones. Thus, functional impairment of anti-apoptotic proteins is
critical for reversion of chemo-resistance.
[0317] The nature of the molecular cues that trigger the aberrant
activation of survival pathways in the clones, hence their altered
phenotype, are unclear at present. In wt Daudi rituximab induces a
rapid and transient increase in A-SMase activity parallel with
cellular CER generation in lipid rafts. These cells externalize
both CER and A-SMase which co-localize with CD20. Also,
rituximab-induced growth inhibition may be mediated through a
CER-dependent pathway (Bezombes, C. et al., Blood, 104:1166-73
(2004)). Preliminary observations suggest rituximab-induced A-SMase
translocation and CER generation at cell surface is reduced in
clones (Jazirehi, A. et al., Proceedings of AACR, 47 (2006)). Since
micro-domains serve as signaling platforms, these data suggest that
rituximab-resistance in clones is, partly, due to faulty
mobilization of the signalling molecules to lipid rafts and a
crippled CER/A-SMase pathway.
[0318] The present findings are the first report on the
establishment of an in vitro model of RR NHL clones which shows
repeated rituximab exposure results in loss of rituximab's ability
to regulate molecular switches leading to constitutive
hyper-activation of survival pathways, over-expression of resistant
factors and increased apoptosis threshold. Accordingly, rituximab
fails to exert anti-lymphoma effects and RR clones develop higher
drug-resistance, which can account for the treatment-refractory and
the aggressive nature of clinical rituximab-/drug-resistant NHL.
FIG. 6 illustrates potential mechanisms of RR. The observed drug-
and rituximab-resistance of RR clones mimic those observed
previously (Olejniczak, S. et al., Blood, 106:4819 (2005)). Though,
RR clones are still amenable to chemotherapy using specific
molecular targeting of the components of deregulated pathways. Our
studies identify several such targets for potential molecular
intervention in the treatment of rituximab-/drug-resistant NHL.
Analysis of dynamics of survival pathways in patient-derived
specimens may also serve as biomarkers in choosing treatment
options. Such patients may be suitable candidates for alternative
(e.g., targeted therapy) over conventional regimens.
[0319] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *
References